Systems and methods for identifying Replikin Scaffolds and uses of said Replikin Scaffolds

ABSTRACT

The present invention provides a new class of peptides related to rapid replication and high human mortality, and their use in diagnosing, preventing and treating disease including vaccines and therapeutics for emerging viral diseases and methods of identifying the new class of peptides and related structures.

This application claims priority to U.S. Provisional Appln. Ser. No.60/653,083, filed Feb. 16, 2005, and is a continuation-in-part of U.S.application Ser. No. 11/116,203, filed Apr. 28, 2005 now U.S. Pat. No.7,774,144, which claims priority to U.S. Provisional Appln. Ser. No.60/565,847, filed Apr. 28, 2004, and is a continuation-in-part of U.S.application Ser. No. 10/860,050, filed Jun. 4, 2004, now U.S. Pat. No.7,442,761. Each of the foregoing applications is incorporated herein byreference in its entirety. The following applications are additionallyincorporated herein by reference: U.S. Provisional Applns. 60/531,686,filed Dec. 23, 2003, 60/504,958, filed Sep. 23, 2003, and 60/476,186,filed Jun. 6, 2003, U.S. application Ser. No. 10/189,437, filed Jul. 8,2002, now U.S. Pat. No. 7,452,963, U.S. application Ser. No. 10/105,232,filed Mar. 26, 2002, now U.S. Pat. No. 7,189,800, U.S. application Ser.No. 09/984,057, filed Oct. 26, 2001, now U.S. Pat. No. 7,420,028, andU.S. Provisional Applns. 60/303,396, filed Jul. 9, 2001, and 60/278,761,filed Mar. 27, 2001.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to two newly discovered classes ofpeptides that share structural characteristics and the use ofbioinformatics to search databases of amino acids, nucleic acids andother biological information to identify shared structuralcharacteristics. Replikins are a newly discovered class of peptides thatshare structural characteristics and have been correlated with rapidreplication of viruses and organisms. Replikin Scaffolds are a sub-setof the class of Replikin peptides. Exoskeleton Scaffolds are anothernewly discovered class of peptides that share structural characteristicsand have been correlated with a decrease in replication.

BACKGROUND OF THE INVENTION

Rapid replication is characteristic of virulence in certain bacteria,viruses and malignancies, but no chemistry common to rapid replicationin different organisms has been described. The inventors have found afamily of conserved small protein sequences related to rapidreplication, Replikins. Such Replikins offer new targets for developingeffective detection methods and therapies. There is a need in the artfor methods of identifying patterns of amino acids such as Replikins.

Bioinformatic Identification of Amino Acid Sequences

Identification of amino acid sequences, nucleic acid sequences and otherbiological structures may be aided with the implementation ofbioinformatics. Publicly available databases containing amino acid andnucleic acid sequence information may be searched to identify and defineReplikins, Replikin Scaffolds and Exoskeleton Scaffolds withinrepresentative proteins or protein fragments or genomes or genomefragments.

Databases of amino acids and proteins are maintained by a variety ofresearch organizations, including, for example, the National Center forBiotechnology Information (NCBI) at the U.S. National Library ofMedicine, and the Influenza Sequence Database at the Los Alamos NationalLaboratory. These databases are typically accessible via the Internetthrough web pages that provide a researcher with capabilities to searchfor and retrieve specific proteins.

Amino Acid Search Tools

As is known in the art, databases of proteins and amino acids may besearched using a variety of database tools and search engines. Usingthese publicly available tools, patterns of amino acids may be describedand located in many different proteins corresponding to many differentorganisms. Several methods and techniques are available by whichpatterns of amino acids may be described. One popular format is thePROSITE pattern. A PROSITE pattern description may be assembledaccording to the following rules:

(1) The standard International Union of Pure and Applied Chemistry(IUPAC) one-letter codes for the amino acids are used (see FIG. 12).

(2) The symbol ‘x’ is used for a position where any amino acid isaccepted.

(3) Ambiguities are indicated by listing the acceptable amino acids fora given position, between square parentheses ‘[ ]’. For example: [ALT]would stand for Alanine or Leucine or Threonine.

(4) Ambiguities are also indicated by listing between a pair of curlybrackets ‘{ }’ the amino acids that are not accepted at a givenposition. For example: {AM} stands for any amino acid except Alanine andMethionine.

(5) Each element in a pattern is separated from its neighbor by a ‘-’.

(6) Repetition of an element of the pattern can be indicated byfollowing that element with a numerical value or a numerical rangebetween parentheses. Examples: x(3) corresponds to x-x-x, x(2,4)corresponds to x-x or x-x-x or x-x-x-x.

(7) When a pattern is restricted to either the N- or C-terminal of asequence, that pattern either starts with a ‘<’ symbol or respectivelyends with a ‘>’ symbol.

(8) A period ends the pattern.

Examples of PROSITE patterns include:

PA [AC]-x-V-x(4)-{ED}. This pattern is translated as: [Alanine orCysteine]-any-Valine-any-any-any-any-{any but Glutamic Acid or AsparticAcid}

PA <A-x-[ST](2)-x(0,1)-V. This pattern, which must be in the N-terminalof the sequence (‘<’), is translated as: Alanine-any-[Serine orThreonine]-[Serine or Threonine]-(any or none)-Valine.

Another popular format for describing amino acid sequence patterns isthe regular expression format that is familiar to computer scientists.In computer science, regular expressions are typically used to describepatterns of characters for which finite automata can be automaticallyconstructed to recognize tokens in a language. Possibly the most notableregular expression search tool is the Unix utility grep.

In the context of describing amino acid sequence patterns, a simplifiedset of regular expression capabilities is typically employed. Amino acidsequence patterns defined by these simple regular expression rules endup looking quite similar to PROSITE patterns, both in appearance and inresult. A regular expression description for an amino acid sequence maybe created according to the following rules:

-   -   (1) Use capital letters for amino acid residues and put a “-”        between two amino acids (not required).    -   (2) Use “[ . . . ]” for a choice of multiple amino acids in a        particular position. [LIVM] means that any one of the amino        acids L, I, V, or M can be in that position.    -   (3) Use “{ . . . }” to exclude amino acids. Thus, {CF} means C        and F should not be in that particular position. In some        systems, the exclusion capability can be specified with a “^”        character. For example, ^G would represent all amino acids        except Glycine, and [^ILMV] would represents all amino acids        except I, L, M, and V.    -   (4) Use “x” or “X” for a position that can be any amino acid.    -   (5) Use “(n)”, where n is a number, for multiple positions. For        example, x(3) is the same as “xxx”.    -   (6) Use “(n1,n2)” for multiple or variable positions. Thus,        x(1,4) represents “x” or “xx” or “xxx” or “xxxx”.    -   (7) Use the symbol “>” at the beginning or end of the pattern to        require the pattern to match the N or C terminus. For example,        “>MDEL” (SEQ ID NO: 13) finds only sequences that start with        MDEL (SEQ ID NO: 13). “DEL>” finds only sequences that end with        DEL.

The regular expression, “[LIVM]-[VIC]-x (2)-G-[DENQTA]-x-[GAC]-x(2)-[LIVMFY](4)-x (2)-G” illustrates a 17 amino acid peptide that has:an L, I, V, or M at position 1; a V, I, or C at position 2; any residueat positions 3 and 4; a G at position 5 and so on . . . .

Other similar formats are in use as well. For example, the Basic LocalAlignment Search Tool (BLAST) is a well-known system available on theInternet, which provides tools for rapid searching of nucleotide andprotein databases. BLAST accepts input sequences in three formats: FASTAsequence format, NCBI Accession numbers, or GenBank sequence numbers.However, these formats are even simpler in structure than regularexpressions or PROSITE patterns. An example sequence in FASTA format is:

>gi|532319|pir|TVFV2E|TVFV2E envelope proteinELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTTGLL LNGSYSENRTQIWQKHRTSNDSALILLNKHYNLTVTCKRPGNKTVLPVTIMAGLVFHSQK YNLRLRQAWCHFPSNWKGAWKEVKEEIVNLPKERYRGTNDPKRIFFQRQWGDPETANLWF NCHGEFFYCKMDWFLNYLNNLTVDADHNECKNTSGTKSGNKRAPGPCVQRTYVACHIRSV IIWLETISKKTYAPPREGHLECTSTVTGMTVELNYIPKNRTNVTLSPQIESIWAAELDRY KLVEITPIGFAPTEVRRYTGGHERQKRVPFVXXXXXXXXXXXXXXXXXXXXXXVQSQHLL AGILQQQKNLLAAVEAQQQMLKLTIWGVK (SEQ ID NO: 15).

Features of the BLAST system include sequence comparison algorithms thatare used to search sequence databases for regions of local alignments inorder to detect relationships among sequences which share regions ofsimilarity. However, the BLAST tools are limited in terms of thestructure of amino acid sequences that can be discovered and located.For example, BLAST is not capable of searching for a sequence that has“at least one lysine residue located six to ten amino acid residues froma second lysine residue,” as required by a Replikin pattern, forexample. Nor is BLAST capable of searching for amino acid sequences thatcontain a specified percentage or concentration of a particular aminoacid, such as a sequence that has “at least 6% lysine residues.”

Need for Replikin Search Tools

As can be seen from its definition, a Replikin pattern descriptioncannot be represented as a single linear sequence of amino acids. Thus,PROSITE patterns and regular expressions, both of which are well suitedto describing ordered strings obtained by following logicalset-constructive operations such as negation, union and concatenation,are inadequate for describing Replikin patterns.

In contrast to linear sequences of amino acids, a Replikin pattern ischaracterized by attributes of amino acids that transcend simplecontiguous ordering. In particular, the requirement that a Replikinpattern contain at least 6% lysine residues, without more, means thatthe actual placement of lysine residues in a Replikin pattern isrelatively unrestricted. Thus, in general, it is not possible torepresent a Replikin pattern description using a single PROSITE patternor a single regular expression.

Accordingly, there is a need in the art for a system and method to scana given amino acid sequence and identify and count all instances of aReplikin pattern. Similarly, there is a need in the art for a system andmethod to search protein databases and amino acid databases for aminoacid sequences that match a Replikin pattern. Additionally, there is aneed in the art for a generalized search tool that permits researchersto locate amino acid sequences of arbitrary specified length thatincludes any desired combination of the following characteristics: (1) afirst amino acid residue located more than N positions and less than Mpositions away from a second amino acid residue; (2) a third amino acidresidue located anywhere in the sequence; and (3) the sequence containsat least R percent of an amino acid residue. Finally, the shortcomingsof the prior art are even more evident in research areas relating todisease prediction and treatment. There is a significant need in the artfor a system to predict in advance the occurrence of disease (forexample, to predict strain-specific influenza epidemics) and similarlyto enable synthetic vaccines to be designed based on amino acidsequences or amino acid motifs that are discovered to be conserved overtime and which have not been previously detectable by prior art methodsof searching proteins and amino acid sequences.

SUMMARY OF THE INVENTION

The present invention provides a method for identifying nucleotide oramino acid sequences that include a Replikin sequence. The method isreferred to herein as a 3-point-recognition method. By use of the“3-point recognition” method, peptides comprising from 7 to about 50amino acids including (1) at least one lysine residue located six to tenamino acid residues from a second lysine residue; (2) at least onehistidine residue; and (3) at least 6% lysine residues and havingreplication, transformation, or redox functions may be identified.

An aspect of the present invention provides a method of identifying aReplikin Scaffold in a virus or organism comprising identifying a seriesof Replikin Scaffold peptides comprising about 16 to about 30 aminoacids comprising (1) a terminal lysine and a lysine immediately adjacentto said terminal lysine; (2) a terminal histidine and a histidineimmediately adjacent to said terminal histidine, (3) a lysine withinabout 6 to about 10 amino acids from another lysine; and (4) at least 6%lysines.

An aspect of the invention may provide a method of identifying aReplikin Scaffold peptide in a virus or organism comprising about 16 toabout 30 amino acids comprising (1) a terminal lysine and a lysineimmediately adjacent to the terminal lysine; (2) a terminal histidineand a histidine immediately adjacent to the terminal histidine, (3) alysine within about 6 to about 10 amino acids from another lysine; and(4) at least 6% lysines.

An aspect of the invention may also provide a method of making apreventive or therapeutic virus vaccine comprising identifying aReplikin Scaffold comprising about 16 to about 30 amino acids andsynthesizing said Replikin Scaffold as a preventive or therapeutic virusvaccine wherein said Replikin Scaffold further comprises: (1) a terminallysine and a lysine immediately adjacent to the terminal lysine; (2) aterminal histidine and a histidine immediately adjacent to the terminalhistidine; (3) a lysine within about 6 to about 10 amino acids fromanother lysine; and (4) at least 6% lysines. The Replikin Scaffold maycontain influenza virus peptide Replikins. A Replikin Scaffold mayfurther comprise a group of Replikins comprising: (1) a terminal lysineand a lysine immediately adjacent to the terminal lysine; (2) a terminalhistidine and a histidine immediately adjacent to the terminalhistidine; (3) a lysine within about 6 to about 10 amino acids fromanother lysine; and (4) at least 6% lysines.

An aspect of the invention may provide a method of identifying anExoskeleton Scaffold wherein a Replikin Scaffold is identified in afirst strain of virus or organism and the Exoskeleton Scaffold isidentified in a later-arising strain of said virus or organism whereinsaid Exoskeleton Scaffold comprises an amino acid sequence comprisingthe same number of amino acids as the Replikin Scaffold and furthercomprising (1) two terminal lysines, (2) two terminal histidines, and(3) no lysine within about 6 to about 10 amino acids from anotherlysine.

In an aspect of the invention an isolated or synthesized influenza viruspeptide is provided with from 7 to about 50 amino acids, at least onelysine residue located six to ten residues from a second lysine residue,at least one histidine residue and at least 6% lysine residues. In afurther aspect the peptide comprises a terminal lysine. In yet a furtheraspect the peptide is present in an emerging strain of influenza virussuch as the influenza virus strain H5N1.

In another aspect of the invention an isolated or synthesized influenzavirus peptide is provided comprising the H5N1 peptideKKNSTYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 15).

In another aspect of the invention, an isolated or synthesized influenzavirus peptide is provided having about 16 to about 30 amino acids; aterminal lysine and a lysine immediately adjacent to the terminallysine; a terminal histidine and a histidine immediately adjacent to theterminal histidine; a lysine within about 6 to about 10 amino acids fromanother lysine; and at least 6% lysines.

In another aspect of the invention, a preventive or therapeutic virusvaccine is provided having at least one isolated or synthesized peptideof influenza virus with at least one lysine residue located six to tenresidues from a second lysine residue; at least one histidine residue;and at least 6% lysine residues. In a further aspect of the inventionthe isolated or synthesized peptide is present in an emerging strain ofinfluenza virus or is present in an H5N1 strain of influenza virus.

In yet a further aspect of the invention, a preventive or therapeuticvirus vaccine comprises the peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHH (SEQID NO: 15) having alternatively a synthetic UTOPE tail, an adjuvant, ora combination thereof. In yet a further aspect, the preventive ortherapeutic virus vaccine comprises a pharmaceutically acceptablecarrier.

In a further aspect of the invention the preventive or therapeutic virusvaccine comprises the peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK(SEQ ID NO: 16)-KLH, where -KLH denotes a key limpet hemocyanin.

In yet another aspect of the invention a method of stimulating theimmune system of a subject to produce antibodies to influenza virus isprovided comprising administering an effective amount of at least oneisolated or synthesized influenza virus Replikin peptide comprising from7 to about 50 amino acids comprising (1) at least one lysine residuelocated six to ten amino acid residues from a second lysine residue; (2)at least one histidine residue; and (3) at least 6% lysine residues.

In a further aspect, in the method of stimulating the immune system theadministered Replikin peptide may further comprise a pharmaceuticallyacceptable carrier and/or adjuvant and prevent or treat an influenzainfection. The method of stimulating the immune system may furthercomprise an isolated or synthesized influenza virus peptide present inan emerging virus or present in an H5N1 strain of influenza virus. Themethod may further comprise administration of the peptideKKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK (SEQ ID NO: 16)-KLH, where-KLH denotes a key limpet hemocyanin.

An aspect of the invention may also provide a method comprising:applying a plurality of criteria to data representing protein sequences;based on the criteria, identifying an arbitrary sub-sequence within theprotein sequences; and outputting the identified sub-sequence to a datafile; wherein the criteria include: a set {a} of amino acids to beincluded in the sub-sequence; a set {b} of amino acids to be excludedfrom the sub-sequence; and a minimum and a maximum permissible gapbetween members of sets {a} and {b}. Within the method the proteinsequences may be obtained via a network. An aspect of the invention mayfurther comprise a machine-readable medium storing computer-executableinstructions to perform such a method.

An aspect of the invention may further provide a method comprisingapplying a plurality of criteria to data representing protein sequences;based on the criteria, identifying a sub-sequence within the proteinsequences, the identified sub-sequence having a predetermined allowedrange of distance between lysine amino acids thereof, and apredetermined allowed range of distance between a histidine amino acidand a farthest Lysine acid thereof; and outputting an identifiedsub-sequence to a data file. The protein sequences may be obtained via anetwork. A machine-readable medium storing computer-executableinstructions may perform such a method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting the frequency of occurrence of Replikinsin various organisms.

FIG. 2 is a graph depicting the percentage of malignin per milligramtotal membrane protein during anaerobic replication of glioblastomacells.

FIG. 3 is a bar graph showing amount of antimalignin antibody producedin response to exposure to the recognin 16-mer (SEQ ID NO: 4).

FIG. 4A is a photograph of a blood smear taken with ordinary andfluorescent light. FIG. 4B is a photograph of a blood smear taken withordinary and fluorescent light illustrating the presence of two leukemiacells. FIG. 4C is a photograph of a dense layer of glioma cells in thepresence of antimalignin antibody. FIG. 4D and FIG. 4E are photographsof the layer of cells in FIG. 4C taken at 30 and 45 minutes followingaddition of antimalignin antibody.

FIG. 4F is a bar graph showing the inhibition of growth of small celllung carcinoma cells in vitro by antimalignin antibody.

FIG. 5 is a plot of the amount of antimalignin antibody present in theserum of patients with benign or malignant breast disease pre- and postsurgery.

FIG. 6 is a box diagram depicting an aspect of the invention wherein acomputer is used to carry out the 3-point-recognition method ofidentifying Replikin sequences.

FIG. 7 is a graph showing the concentration of Replikins observed inhemagglutinin of influenza B and influenza A strain, H1N1, on a year byyear basis from 1940 through 2001.

FIG. 8 is a graph of the Replikin concentration observed inhemagglutinin of influenza A strains, H2N2 and H3N2, as well as anemerging strain defined by its constituent Replikins, designatedH3N2(R), on a year by year basis from 1950 to 2001.

FIG. 9 is a graph depicting the Replikin count per year for severalvirus strains, including the coronavirus nucleocapsid Replikin, from1917 to 2002.

FIG. 10, which contains FIGS. 10A, 10B, and 10C, is a chart depictingthe mean Replikin count per year for nucleocapsid coronavirus isolates.FIG. 10A depicts years 1962 to 1978. FIG. 10B depicts years 1979 to1992. FIG. 10C depicts years 1993 to 2003.

FIG. 11 is a chart depicting the Replikin count per year for H5N1Hemagglutinins.

FIG. 12 is a conversion table that enables amino acids to be encoded assingle alphabetic characters according to a standard supplied by theInternational Union of Pure and Applied Chemistry (IUPAC).

FIG. 13 is a printout of a human cancer protein (SEQ ID NO: 472)obtained by searching a protein database maintained by the NationalCenter for Biotechnology Information (NCBI).

FIG. 14 is a conversion table illustrating a correspondence betweennucleic acid base triplets and amino acids.

FIG. 15 is a graph illustrating a rapid increase in the concentration ofReplikin patterns in the hemagglutinin protein of the H5N1 strain ofinfluenza prior to the outbreak of three “Bird Flu” epidemics. FIG. 15illustrates that increasing replikin concentration (‘Replikin Count’) ofhemagglutinin protein of H5N1 preceded three ‘Bird Flu’ Epidemics. InH5N1 influenza, the increasing strain-specific replikin concentration(Replikin Count, Means±/−SD) 1995 to 1997 preceded the Hong Kong H5N1epidemic of 1997 (E1); the increase from 1999 to 2001 preceded theepidemic of 2001 (E2); and the increase from 2002 to 2004 preceded theepidemic in 2004 (E3). The decline in 1999 occurred with the massiveculling of poultry in response to the E1 epidemic in Hong Kong.

FIG. 16 is a table illustrating selected examples of Replikin patternsthat have been found in various organisms.

FIG. 17 is a high-level block diagram of a computer system incorporatinga system and method for identifying Replikin patterns in amino acidsequences, in accordance with an aspect of the present invention.

FIG. 18 is a simple flow chart illustrating a general method forlocating a Replikin pattern in a sequence of amino acids, according toan aspect of the present invention.

FIG. 19 is a flow chart illustrating a generalized method for locating aplurality of Replikin-like patterns in a sequence of amino acids,according to an aspect of the present invention.

FIG. 20 is a source code listing containing a procedure for discoveringReplikin patterns in a sequence of amino acids, in accordance with anaspect of the present invention.

FIG. 21 is a table illustrating Replikin Scaffolds occurring insubstantially fixed amino acid positions in different proteins. Figurediscloses SEQ ID NOS: 473-531, respectively, in order of appearance.

FIG. 22 is a simplified block diagram of a computer system platformuseful with the present invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “peptide” or “protein” refers to a compound oftwo or more amino acids in which the carboxyl group of one is unitedwith an amino group of another, forming a peptide bond. The term peptideis also used to denote the amino acid sequence encoding such a compound.As used herein, “isolated” or “synthesized” peptide or biologicallyactive portion thereof refers to a peptide that is after purificationsubstantially free of cellular material or other contaminating proteinsor peptides from the cell or tissue source from which the peptide isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized by any method, or substantiallyfree from contaminating peptides when synthesized by recombinant genetechniques.

As used herein, a Replikin peptide or Replikin protein is an amino acidsequence having 7 to about 50 amino acids comprising:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue;    -   (3) at least 6% lysine residues.        Similarly, a Replikin sequence is the amino acid sequence        encoding such a peptide or protein.

As used herein, an “earlier-arising” virus or organism is a specimen ofa virus or organism collected from a natural source of the virus ororganism on a date prior to the date on which another specimen of thevirus or organism was collected. A “later-arising” virus or organism isa specimen of a virus or organism collected from a natural source of thevirus or organism on a date subsequent to the date on which anotherspecimen of the virus or organism was collected.

As used herein, “emerging strain” as used herein refers to a strain of avirus, bacterium, fungus, or other organism identified as having anincreased increasing concentration of Replikin sequences in one or moreof its protein sequences relative to the concentration of Replikins inother strains of such organism. The increase or increasing concentrationof Replikins occurs over a period of at least about six months, andpreferably over a period of at least about one year, most preferablyover a period of at least about three years or more, for example, ininfluenza virus, but may be a much shorter period of time for bacteriaand other organisms.

As used herein, “mutation” refers to change in this structure andproperties of an organism caused by substitution of amino acids. Incontrast, the term “conservation” as used herein, refers to conservationof particular amino acids due to lack of substitution.

As used herein, “replikin count” refers to the number of replikins per100 amino acids in a protein or organism. A higher replikin count in afirst strain of virus or organism has been found to correlate with morerapid replication of the first virus or organism as compared to asecond, earlier- or later-arising strain of the virus or organism havinga lower replikin count.

As used herein “Replikin Scaffold” refers to a series of conservedReplikin peptides wherein each of said Replikin peptide sequencescomprises about 16 to about 30 amino acids and further comprises: (1) aterminal lysine; (2) a terminal histidine and a histidine immediatelyadjacent to the terminal histidine; (3) a lysine within 6 to 10 aminoacid residues from another lysine; and (4) about 6% lysine. “ReplikinScaffold” peptides may comprise an additional lysine immediatelyadjacent to the terminal lysine. “Replikin Scaffold” also refers to anindividual member or a plurality of members of a series of a “ReplikinScaffold.”

Identification of Replikins

The identification of a new family of small peptides related to thephenomenon of rapid replication, referred to herein as Replikins,provides targets for detection of pathogens in a sample and developingtherapies, including vaccine development. In general, knowledge of andidentification of this family of peptides enables development ofeffective therapies and vaccines for any organism that harborsReplikins. Identification of this family of peptides also provides forthe detection of viruses and virus vaccine development.

For example, identification of this family of peptides provides for thedetection of influenza virus and provides new targets for influenzatreatment and vaccines including treatment and vaccines for influenzaH5N1. Further examples provided by the identification of this family ofpeptides include the detection of infectious disease Replikins, cancerimmune Replikins and structural protein Replikins.

Rapid replication is characteristic of virulence in certain bacteria,viruses and malignancies, but no chemistry common to rapid replicationin different organisms has been described. We have found a family ofconserved small protein sequences related to rapid replication, which wehave named Replikins. Such Replikins offer new targets for developingeffective detection methods and therapies. The first Replikin found wasthe glioma Replikin, which was identified in brain glioblastomamultiforme (glioma) cell protein, called malignin.

Hydrolysis and mass spectrometry of malignin revealed the novel 16-merpeptide sequence which contains the glioma Replikin. This Replikin wasnot found in databases for the normal healthy human genome and thereforeappeared to be derived from some source outside the body.

We have devised an algorithm to search for the glioma Replikin orhomologue thereof. Homologues were not common in over 4,000 proteinsequences, but were found, surprisingly, in all tumor viruses, and inthe replicating proteins of algae, plants, fungi, viruses and bacteria.

We have identified that both 1) Replikin concentration (number ofReplikins per 100 amino acids) and 2) Replikin composition correlatewith the functional phenomenon of rapid replication. These relationshipsprovide functional basis for the determination that Replikins arerelated quantitatively as well as qualitatively to the rate ofreplication.

The first functional basis for Replikins role to rapid replication wasdiscovered by the Applicants in glioma replication. The fact that gliomamalignin was found to be enriched ten-fold compared to the five-foldincrease in cell number and membrane protein concentration in rapidreplication of glioma cells suggests an integral relationship of theReplikins to replication. When the glioma Replikin was synthesized invitro and administered as a synthetic vaccine to rabbits, abundantantimalignin antibody was produced. This establishes the antigenic basisof the antimalignin antibody in serum (AMAS) test, and provides thefirst potential synthetic cancer vaccine and the prototype for Replikinvaccines in other organisms. With the demonstration of this naturalimmune relationship of the Replikins to replication and this naturalimmune response to cancer Replikins, which overrides cell type, basedupon the shared specificity of cancer Replikins and rapid replication,both passive augmentation of this immunity with antimalignin antibodyand active augmentation with synthetic Replikin vaccines now ispossible.

The relationship between the presence of antimalignin antibody andsurvival in patients was shown in a study of 8,090 serum specimens fromcancer patients. The study showed that the concentration of antimaligninantibody increases with age, as the incidence of cancer in thepopulation increases, and increases further two to three-fold in earlymalignancy, regardless of cell type. In vitro, the antimalignin antibodyis cytotoxic to cancer cells at picograms (femtomoles) per cancer cell,and in vivo the concentration of antimalignin antibody relatesquantitatively to the survival of cancer patients. As shown in gliomacells, the stage in cancer at which cells have only been transformed tothe immortal malignant state but remain quiescent or dormant, now can bedistinguished from the more active life-threatening replicating state,which is characterized by the increased concentration of Replikins. Inaddition, clues to the viral pathogenesis of cancer may be found in thefact that glioma glycoprotein 10B has a 50% reduction in carbohydrateresidues when compared to the normal 1OB. This reduction is associatedwith virus entry in other instances, and so may be evidence of theattachment of virus for the delivery of virus Replikins to the 10B ofglial cells as a step in the transformation to the malignant state.

Our study concerning influenza virus hemagglutinin protein sequences andinfluenza epidemiology over the past 100 years has provided a secondfunctional basis for the relations of Replikins to rapid replication.Only serological hemagglutinin and antibody classification, but nostrain-specific conserved peptide sequences have previously beendescribed in influenza. Further, no changes in concentration andcomposition of any strain-specific peptide sequences have been describedpreviously that correlate with epidemiologically documented epidemics orrapid replication. In this study, a four to ten-fold increase in theconcentration of strain-specific influenza Replikins in one of each ofthe four major strains, influenza B, (A)H1N1, (A)H2N2 and, (A)H3N2 isshown to relate to influenza epidemics caused by each strain from 1902to 2001.

We then showed that these increases in concentration are due to thereappearance of at least one specific Replikin composition from 1 to upto 64 years after its disappearance, plus the emergence of newstrain-specific Replikin compositions. Previously, no strain-specificchemical structures were known with which to predict the strains thatwould predominate in coming influenza seasons, nor to devise annualmixtures of whole-virus strains for vaccines. The recent sharp increasein H3N2 Replikin concentration (1997 to 2000), the largest in H3N2'shistory, and the reappearance of specific Replikin compositions thatwere last seen in the high mortality H3N2 pandemic of 1968, and in thetwo high mortality epidemics of 1975 and 1977, but were absent for 20-25years, together may be a warning of coming epidemics. This high degreeof conservation of Replikin structures observed, whereby the identicalstructure can persist for 100 years, or reappear after an absence offrom one to 64 years, indicate that what was previously thought to bechange due to random substitution of amino acids in influenza proteinsis more likely to be change due to an organized process of conservationof Replikins.

The conservation of Replikins is not unique to influenza virus but wasalso observed in other sources, for example in foot and mouth diseasevirus, type 0, HIV tat, and wheat.

A third functional basis for Replikins' role in rapid replication isseen in the increase in rapid replication in HIV. Replikin concentrationwas shown to be related to rapid replication in HIV. We found theReplikin concentration in the slow growing low-titre strain of HIV (NS1,“Bru”), which is prevalent in early stage infection, to be one-sixth ofthe Replikin concentration in the rapidly-growing high-titre strain ofHIV (SI, “Lai”)(prevalent in late stage HIV infection).

Further examples demonstrate the relationship of Replikins to rapidreplication. In the “replicating protein,” of tomato leaf curl geminivirus, which devastates tomato crops, the first 161 amino acids, thesequence that has been shown to bind to DNA, was shown to contain fiveReplikins. In malaria, legendary for rapid replication when trypanosomesare released from the liver in the tens of thousands from onetrypanosome, multiple, novel, almost ‘flamboyant’ Replikin structureshave been found with concentrations of up to 36 overlapping Replikinsper 100 amino acids.

The conservation of any structure is critical to whether that structureprovides a stable invariant target to attack and destroy or tostimulate. When a structure is tied in some way to a basic survivalmechanism of the organism, the structures tend to be conserved. Avarying structure provides an inconstant target, which is a goodstrategy for avoiding attackers, such as antibodies that have beengenerated specifically against the prior structure and thus areineffective against the modified form. This strategy is used byinfluenza virus, for example, so that a previous vaccine may be quiteineffective against the current virulent virus.

Replikins as Stable Targets for Treatment

Both bacteria and HIV have both Replikin and non-Replikin amino acids.In HIV, for example, there has been a recent increase in drug-resistancefrom 9% to 13% due to mutation, that is, substitution of amino acids notessential to the definition of the Replikin structure. (See detailedanalysis of TAT protein of HIV discussed herein). In bacteria, thedevelopment of ‘resistant strains’ is due to a similar mechanism.However, we have found that Replikin structures do not mutate or changeto the same degree as non Replikin amino acids (see also discussion offoot and mouth disease virus conservation of Replikins discussed herein;further see discussion of conservation of coronavirus Replikinsdiscussed herein). The Replikin structures, as opposed to thenon-Replikin structures are conserved and thus provide new constanttargets for treatment.

Certain structures too closely related to survival functions apparentlycannot change constantly. Because an essential component of the Replikinstructure is histidine (h), which is know for its frequent binding tometal groups in redox enzymes and probable source of energy needed forreplication, and since this histidine structure remains constant, thisstructure remains all the more attractive a target for destruction orstimulation.

From a proteomic point of view, the inventors' construction of atemplate based on the newly determined glioma peptide sequence led themto the discovery of a wide class of proteins with related conservedstructures and a particular function, in this case replication. Examplesof the increase in Replikin concentration with virulence of a diseaseinclude, influenza, HIV, cancer and tomato leaf curl virus. This newlyrecognized class of structures is related to the phenomenon of rapidreplication in organisms as diverse as influenza, yeast, algae, plants,the gemini curl leaf tomato virus, HIV and cancer.

Replikin concentration and composition provide new quantitative methodsto detect and control the process of replication, which is central tothe survival and dominance of each biological population. The sharing ofimmunological specificity by diverse members of the class, asdemonstrated with antimalignin antibody for the glioma and relatedcancer Replikins, suggests that B cells and their product antibodies mayrecognize Replikins by means of a similar recognition language.

Examples of peptide sequences of cancer Replikins or as containing aReplikin, i.e., a homologue of the glioma peptide, kagvaflhkk (SEQ IDNO: 1), may be found in such cancers of, but not limited to, the lung,brain, liver, soft-tissue, salivary gland, nasopharynx, esophagus,stomach, colon, rectum, gallbladder, breast, prostate, uterus, cervix,bladder, eye, forms of melanoma, lymphoma, leukemia, and kidney.

Replikins provide for: 1) detection of pathogens by qualitative andquantitative determinations of Replikins; 2) treatment and control of abroad range of diseases in which rapid replication is a key factor bytargeting native Replikins and by using synthetic Replikins as vaccines;and 3) fostering increased growth rates of algal and plant foods.

The first Replikin sequence to be identified was the cancer cellReplikin found in a brain cancer protein, malignin, which wasdemonstrated to be enriched ten-fold during rapid anaerobic replicationof glioblastoma multiforme (glioma) cells. (FIG. 2) Malignin is a 10 KDaportion of the 250 KDa glycoprotein 10B, which was isolated in vivo andin vitro from membranes of glioblastoma multiforme (glioma) cells.Hydrolysis and mass spectroscopy of malignin revealed a 16-mer peptidesequence, ykagvaflhkkndide (SEQ ID NO:4), which is referred to herein asthe glioma Replikin and which includes the shorter peptide, kagvaflhkk(SEQ ID NO: 1), both of which apparently are absent in the normal humangenome.

TABLE 1 16-mer peptide sequence YKAGVAFLHKKNDIDE (SEQ ID NO: 4) obtainedfrom malignin by hydrolysis and mass spectrometry Method By WhichFragment Obtained Auto- Auto- hydrolysis of hydrolysis malignin ofimmobilized Seq malignin on ID Fragment MH+ free in bromoacetylMicrowaved Microwaved NO. Identified (mass) Sequence solution cellulose5 seconds 30 seconds 19 1-3 381.21 ( )yka(g) + 20 1-5 537.30 ()ykagv(a) + 21 2-6 445.28 (y)kagva(f) + 22 2-7 592.35 (y)kagvaf(l) + 23 4-11 899.55 (a)gvaflhkk(n) + 24 5-7 336.19 (g)vaf(l) + 25 6-7 237.12(v)af(l) + 26  6-10 615.36 (v)aflhk(k) + 27  6-10 615.36 (v)aflhk(k) +28  6-12 857.50 (v)aflhkkn(d) + 29  6-12 857.50 (v)afhkkn(d) + 30 7-8279.17 (a)fl(h) + 31 10-16 861.43 (h)kkndide( ) + 32 11-14 489.27(k)kndi(d) + 33 12-15 476.2- (k)ndid(e) +

When the 16-mer glioma Replikin was synthesized and injected as asynthetic vaccine into rabbits, abundant antimalignin antibody wasproduced. (Bogoch et al., Cancer Detection and Prevention, 26 (Suppl.1): 402 (2002)). The concentration of antimalignin antibody in serum invivo has been shown to relate quantitatively to the survival of cancerpatients. (Bogoch et al., Protides of Biological Fluids, 31:739-747(1984). In vitro antimalignin antibodies have been shown to be cytotoxicto cancer cells at a concentration of picograms (femtomolar) per cancercell. (Bogoch et al., Cancer Detection and Prevention, 26 (Suppl. 1):402 (2002).

Studies carried out by the inventors showed that the glioma Replikin isnot represented in the normal healthy human genome. Consequently, asearch for the origin and possible homologues of the Replikin sequencewas undertaken by analysis of published sequences of various organisms.

By using the 16-mer glioma Replikin sequence as a template andconstructing a recognition proteomic system to visually scan the aminoacid sequences of proteins of several different organisms, a new classof peptides, the Replikins, was identified. The present inventionprovides a method for identifying nucleotide or amino acid sequencesthat include a Replikin sequence. The method is referred to herein as a3-point-recognition method. The three point recognition methodcomprises: a peptide from 7 to about 50 amino acids including (1) atleast one lysine residue located six to ten amino acid residues from asecond lysine residue; (2) at least one histidine residue; and (3) atleast 6% lysine residues. (Replikin). These peptides or proteinsconstitute a new class of peptides in species including algae, yeast,fungi, amoebae, bacteria, plant, virus and cancer proteins havingreplication, transformation, or redox functions. Replikin peptides havebeen found to be concentrated in larger ‘replicating’ and ‘transforming’proteins (so designated by their investigators, See Table 2) and cancercell proteins. No sequences were found to be identical to the malignin16-mer peptide.

The present invention further provides a method for identifyingnucleotide or amino acid sequences that include a Replikin sequencecomprising from 7 to about 50 amino acids including (1) at least onefirst lysine located at either terminus of the isolated or synthesizedpeptide, (2) a second lysine located six to ten residues from the firstlysine residue; (3) at least one histidine; and (4) at least 6% lysines.In another aspect of the invention the isolated or synthesized peptidesare influenza virus peptides. In yet another aspect of the invention,the isolated or synthesized peptides are H5N1 influenza virus peptides.

TABLE 2 Examples of Replikins in various organisms - prototype: Glioma Replikin* KAGVAFLHKK (SEQ ID NO: 1) Algae:  SEQ ID NO:  34Caldophera prolifera kaskftkh 35 Isolepisprolifera kaqaetgeikgh Yeast: 36 Schizosaccharomyces pombe ksfkypkkhk 37 Oryza sativa kkaygnelhk 2Sacch. cerevisiae replication binding protein hsikrelgiifdk Fungi:  38Isocitrate lyase ICI 1,Penicillium marneffei kvdivthqk 39DNA-dependent RNA polymerase 11, Diseula destructiva kleedaayhrkk 40Ophiostoma novo-ulm 1,RNA in Dutch elm disease kvilplrgnikgiffkh fungusAmoeba:  41 Entamoeba invadens, histone H2B klilkgdlnkh Bacteria:  42Pribosomal protein replication factor, Helicobacter pylori ksvhaflkReplication-associated protein Staph. aureus 10Mycoplasma pulmonic, chromosome replication kkektthnk 43Macrophage infectivity potentiator, L. legionella kvhffqlkk 90Bacillus anthracis kihlisvkk 91 Bacillus anthracis hvkkekeknk 92Bacillus anthracis khivkievk 93 Bacillus anthracis kkkkikdiygkdallh 94Bacillus anthracis kwekikqh 95 Bacillus anthracis kklqipppiepkkddiih 96Bacillus anthracis hnryasnivesayllilnew- knniqsdlikk 97Bacillus anthracis havddyagylldknqsdlv- tnskk 98 Bacillus anthracishaerlkvgknapk Plants:  44 Arabidopsis thaliana, prolifera kdhdfdgdk 45Arabidopsis thaliana, cytoplasmic ribosomal kmkglkqkkah 46Arabidopsis thaliana, DNA binding protein kelssttqeksh Viruses:  9Replication associated protein A [Maize streak virus] kekkpskdeimrdiish11 Bovine herpes virus 4, DNA replication protein hkinitngqk 12Meleagrid herpesvirus 1, replication binding protein hkdlyrllmk 47Feline immunodeficiency hlkdyklvk 3 Foot and Mouth Disease (O)hkqkivapvk 5 HIV Type 1 kcfncgkegh 7 HIV Type 2 kcwncgkegh 99Small Pox Virus (Variola) khynnitwyk 100 Small Pox Virus (Variola)kysqtgkeliih 101 Small Pox Virus (Variola) hyddvrikndivvsrck 102Small Pox Virus (Variola) hrfklildski 103 Small Pox Virus (Variola)kerghnyyfek Tumor 48 Rous sarcoma virus tyrosine-protein kinase kklrhekViruses:  49 v-yes, avian sarcoma kklrhdk 50c-yes, colon cancer, malignant melanoma kklrhdk 51 v-srcC, avian sarcomakklrhek 52 c-src, colon, mammary, panrcreatic cancer kklrhek 53Neuroblastoma RAS viral (v-ras) oncogene kqahelak 54VPI (major capsid protein) [Polyamavirus sp.] kthrfskh 55 Sindbisknlhekik 56 E1 [Human papilloamavirus type 71] khrpllqlk 57v-erbB from AEV and c-erb kspnhvk 58 v-fms (feline sarcoma) knihlekk 59c-fms (acute and chronic myelomonocytic tumors) knihlekk 60large t-antigen I [Polyomavirus sp.1 kphlaqslek 61middle t-antigen [Polyomavirus sp,1- kqhrelkdk 62small t-antigen [Polyomavirus spJ, kqhrelkdk 63v-abl, murine acute leukemia kvpvlisptlkh 64Human T-cell lymphotropic virus typo 2 kslllevdkdish 65c-kit, GI tumors, small cell lung carcinoma kagitimvkreyh 18 Hepatitis Chyppkpgcivpak Trans- 66 Transforming protein myb ksgkhlgk Forming 67Transforming protein myc, Burkitt lymphoma krreqlkhk Proteins:  68Ras-related GTP-binding protein ksfevikvih 69Transforming protein ras (teratocarcinoma) kkkhtvkk 70TRAF-associated NF · kB activator TANK kaqkdhlsk 71RFP transforming protein hlkrvkdlkk 72 Transforming protein D (S.C.)kygspkhrlik 73 Papilloma virus type 11, transforming proteinklkhilgkarfik 74 Protein tryosine kinasc (EC 2.7.1.ll2slk kgdhvkhykirk75 Transforming protein (axl(-)) keklrdvmvdrhk 76Transforming protein (N-myc) klqarqqqllkkieh 77Fibroblast growth factor 4 (Kaposi sarcoma) kkgnrvsptmkvth Cancer 78Matrix metaloproteinase 7 (uterine) keiplhfrk Cell 79Transcription factor 7-like kkkphikk Proteins:  80Breast cancer antigen NY-BR-87 ktrhdplak 81BRCA-1-Associated Ring Domain Protein (breast) khhpkdnlik 82‘Autoantigen from a breast tumor’ khkrkkfrqk 83Glioma Replikin (this study) kagvaflhkk 84 Ovarian cancer antigenkhkrkkfrqk 85 EE L leukemia kkkskkhkdk 86Proto-oncogene tyrosine-protein kinase C-ABLE hksekpalprk 87Adenomatosis polyposis coli kkkkpsrlkgdnek 88Gastric cancer transforming protein ktkkgnrvsptmkvth 89Transforming protein (K-RAS 2B),lung khkekmskdgkkkkkksk

Identification of an amino acid sequence as a Replikin or as containinga Replikin, i.e., a homologue of the glioma peptide, kagvaflhkk (SEQ IDNO: 1), requires that the three following requirements be met. Accordingto the three point recognition system the sequences have three elements:(1) at least one lysine residue located six to ten residues from anotherlysine residue; (2) at least one histidine residue; and (3) acomposition of at least 6% lysine within an amino acid sequence of 7 toabout 50 residues. An exemplary non-limiting Replikin comprises aterminal lysine.

Databases were searched using the National Library of Medicine keyword“PubMed” descriptor for protein sequences containing Replikin sequences.Over 4,000 protein sequences were visually examined for homologues.Sequences of all individual proteins within each group ofPubMed-classified proteins were visually scanned for peptides meetingthe three above-listed requirements. An infrequent occurrence ofhomologues was observed in “virus peptides” as a whole (1.5%) (N=953),and in other peptides not designated as associated with malignanttransformation or replication such as “brain peptides” and“neuropeptides” (together 8.5%) (N=845). However, surprisingly,homologues were significantly more frequently identified in large“replicating proteins,” which were identified as having an establishedfunction in replication in bacteria, algae, and viruses. Even moresurprising was the finding that Replikin homologues occurred in 100% of“tumor viruses” (N=250), in 97% of “cancer proteins” (N=401), and in 85%of “transforming viruses” (N=248). These results suggest that there areshared properties of cancer pathogenesis regardless of cell type andsuggest a role of viruses in carcinogenesis, i.e., conversion of cellsfrom a transformed albeit dormant state to a more virulent activelyreplicating state.

Homologues of the following amino acid sequence, kagvaflhkk (SEQ ID NO:1), as defined by the three point recognition method, were found in suchviruses, or viral peptides, as, but not limited to, adenovirus,lentivirus, a-virus, retrovirus, adeno-associated virus, humanimmunodeficiency virus, hepatitis virus, influenza virus, maize streakvirus, herpes virus, bovine herpes virus, feline immunodeficiency virus,foot and mouth disease virus, small pox virus, rous sarcoma virus,neuroblastoma RAS viral oncogene, polyomavirus, sindbis, human papillomavirus, myelomonocytic tumor virus, murine acute leukemia, T-celllymphotrophic virus, and tomato leaf curl virus.

Furthermore, homologues of the amino acid sequence kagvaflhkk (SEQ IDNO: 1) are present in known classes of coronavirus, which are members ofa family of enveloped viruses that replicate in the cytoplasm of hostcells. Additionally, the homologue of the amino acid sequence kagvaflhkk(SEQ ID NO: 1) is present in the recently identified class ofcoronavirus responsible for severe acute respiratory syndrome, or SARS.The replikin is located in the nucleocapsid whole protein sequence ofthe SARS coronavirus. In addition, the location of the replikins ispresent in other members of the coronavirus class and, morespecifically, are also present in the nucleocapsid protein sequencesfrom these cornaviruses.

Replikins are present in such bacteria as, but not limited to,Acetobacteri, Achromobacter, Actinomyces, Aerobacter, Alcaligenes,Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Chainia,Clostridium, Corynebacterium, Erwinia, Escheria, Lebsiella,Lactobacillus, Haemophilus, Flavobacterium, Methylomonas, Micrococcus,Mycobacterium, Micronomspora, Mycoplasma, Neisseria, Nocardia, Proteus,Pseudomonas, Rhizobium, Salmonella, Serratia, Staphylococcus,Streptocossus, Streptomyces, Streptosporangium, Strepto-virticillium,Vibrio peptide, and Xanthomas. Replikins are present in such fungi as,but not limited to, Penicillium, Diseula, Ophiostoma novo-ulim,Mycophycophta, Phytophthora infestans, Absidia, Aspergillus, Candida,Cephalosporium, Fusarium, Hansenula, Mucor, Paecilomyces, Pichia,Rhizopus, Torulopsis, Trichoderma, and Erysiphe. Replikins are presentin such yeast as, but not limited to, Saccharomyces, Cryptococcus,including Cryptococcusneoformas, Schizo-saccharomyces, and Oryza.Replikins are present in algae such as, but not limited to, Caldophera,Isolepisprolifera, Chondrus, Gracilaria, Gelidium, Caulerpa, Laurencia,Cladophexa, Sargassum, Penicillos, Halimeda, Laminaria, Fucus,Ascophyllum, Undari, Rhodymenia, Macrocystis, Eucheuma, Ahnfeltia, andPteroclasia. Replikins are present in amoeba such as, but not limitedto, Entamoeba (including Entamoeba invadens), Amoebidae, Acanthamoebaand Naegleria. Replikins are present in plants such as, but not limitedto, Arabidopsis, wheat, rice, and maize.

Auxiliary Specifications

To permit classification of subtypes of Replikins, additional or“auxiliary specifications” to the basic “3-point-recognition”requirements may be added: (a) on a structural basis, such as the commonoccurrence of adjacent di- and polylysines in cancer cell proteins(e.g., transforming protein P21B(K-RAS 2B), lung, Table 2, SEQ ID NO:89), and other adjacent di-amino acids in TOLL-like receptors, or b) ona functional basis, such as exhibiting ATPase, tyrosine kinase or redoxactivity as seen in Table 2.

Functional Derivatives

“Functional derivatives” of the Replikins as described herein arefragments, variants, analogs, or chemical derivatives of the Replikins,which retain at least a portion of the immunological cross reactivitywith an antibody specific for the Replikin. A fragment of the Replikinpeptide refers to any subset of the molecule. Variant peptides may bemade by direct chemical synthesis, for example, using methods well knownin the art. An analog of a Replikin to a non-natural proteinsubstantially similar to either the entire protein or a fragmentthereof. Chemical derivatives of a Replikin contain additional chemicalmoieties not normally a part of the peptide or peptide fragment.

Replikins and Replication

As seen in FIG. 2, during anaerobic respiration when the rate of cellreplication is increased, malignin is enriched. That is, malignin isfound to increase not simply in proportion to the increase in cellnumber and total membrane proteins, but is enriched as much as ten-foldin concentration, starting with 3% at rest and reaching 30% of totalmembrane protein. This clear demonstration of a marked increase inReplikin concentration with glioma cell replication points to, and isconsistent with, the presence of Replikins identified with the 3-pointrecognition method in various organisms. For example, Replikins wereidentified in such proteins as “Saccharomyces cerevisiae replicationbinding protein” (SEQ ID NO: 2) (hsikrelgiifdk); the “replicationassociated protein A of maize streak virus” (SEQ ID NO: 8) (kyivcareahk)and (SEQ ID NO: 9) (kekkpskdeimrdiish); the “replication-associatedprotein of Staphylococcus aureus” (SEQ ID NO: 10) (kkektthnk); the “DNAreplication protein of bovine herpes virus 4” (SEQ ID NO: 11)(hkinitngqk); and the “Mealigrid herpes virus 1 replication bindingprotein” (SEQ ID NO: 12) (hkdlyrllmk). Previous studies of tomato leafcurl gemini virus show that the regulation of virus accumulation appearsto involve binding of amino acids 1-160 of the “replicating protein” ofthat virus to leaf DNA and to other replication protein molecules duringvirus replication. Analysis of this sequence showed that amino acids1-135 of this “replicating protein” contain a replikin count(concentration) as high as 20.7 (see section on tomato leaf curl Geminivirus.)

Table 2 shows that Replikin-containing proteins also are associatedfrequently with redox functions, and protein synthesis or elongation, aswell as with cell replication. The association with metal-based redoxfunctions, the enrichment of the Replikin-containing glioma maligninconcentration during anaerobic replication, and the cytotoxicity ofantimalignin at low concentrations (picograms/cell) (FIG. 4C-4F), allsuggest that the Replikins are related to central respiratory survivalfunctions, have been found less often subjected to the mutationscharacteristic of non-Replikin amino acids.

Replikins in Influenza Epidemics

Of particular interest, it was observed that at least one Replikin per100 amino acids was found to be present in the hemagglutinin proteins ofalmost all of the individual strains of influenza viruses examined. TheReplikin sequences that were observed to occur in the hemagglutininproteins of isolates of each of the four prevalent strains of influenzavirus, influenza B, H1N1, H2N2, and H3N2, for each year that amino acidsequence data are available (1902-2001), are shown in Tables 3, 4, 5 and6.

Both the concentration and type, i.e., composition of Replikinsobserved, were found to relate to the occurrence of influenza pandemicsand epidemics. The concentration of Replikins in influenza viruses wasexamined by visually scanning the hemagglutinin amino acid sequencespublished in the National Library of Medicine “PubMed” data base forinfluenza strains isolated world wide from human and animal reservoirsyear by year over the past century, i.e., 1900 to 2001. These Replikinconcentrations (number of Replikins per 100 amino acids, mean±SD) werethen plotted for each strain.

The concentration of Replikins was found to directly relate to theoccurrence of influenza pandemics and epidemics. The concentration ofReplikins found in influenza B hemagglutinin and influenza A strain,H1N1, is shown in FIG. 7, and the concentration of Replikins found inthe two other common influenza virus A strains, H2N2 and H3N2 is shownin FIG. 8 (H2N2, H3N2). The data in FIG. 8 also demonstrate an emergingnew strain of influenza virus as defined by its constituent Replikins(H3N2(R)).

Each influenza A strain has been responsible for one pandemic: in 1918,1957, and 1968, respectively. The data in FIGS. 7 and 8 show that atleast one Replikin per 100 amino acids is present in each of theinfluenza hemagglutinin proteins of all isolates of the four commoninfluenza viruses examined, suggesting a function for Replikins in themaintenance of survival levels of replication. In the 1990s, during thedecline of the H3N2 strain, there were no Replikins in many isolates ofH3N2, but a high concentration of new Replikins appeared in H3N2isolates, which define the emergence of the H3N2(R) strain. See Tables3, 4, 5 and 6.

TABLE 3Replikin Sequences present in hemagglutinins of Influenza B viruses ineach year for which ammo acid sequences were available (1940–2001).Influenza B Replikins Year Detected in Influenza B strainPeak in FIG. 7: E kshfanlk1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 (SEQ ID NO: 104)kshfanlkgtk 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01(SEQ ID NO: 105) kshfanlkgtktrgklcpk1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 (SEQ ID NO: 106)hekygglnk 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01(SEQ ID NO: 107) hekygglnksk1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 (SEQ ID NO: 108)hekygglnkskpyytgehak 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01(SEQ ID NO: 109) hakaigncpiwvk1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 (SEQ ID NO: 110)hakaigncpiwvktplklangtk 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01(SEQ ID NO: 111) hakaigncpiwvktplklangtkyrppak1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 (SEQ ID NO: 112)hakaigncpiwvktplklangtkyrppallk1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 (SEQ ID NO: 113)k(a/v)silhevk 1940,      59,            90,93 (SEQ ID NO: 119)kvwcasgrskvikgslpligeadclh 1940,43,   59,75,76,77,89,90,      98,99,00(SEQ ID NO: 123) kpyytgehak1940,      59,         89,90,93,97,98,      01 (SEQ ID NO: 124)hgvavaadlkstqeaink 1940,      59,                           00(SEQ ID NO: 128) hgvavaadlkstqeainkdtistqeaink 1940 (SEQ ID NO: 129)hsdneiqmvklygdsk (SEQ ID NO: 116) hsdneiqdkmvklygdskpqk (SEQ ID NO: 117)kygglnkskpyytgeh (SEQ ID NO: 122) kcmgtipsakasilhevk   1943,      75,76,77,      93 (SEQ ID NO: 125) klygdskpqkftssangvtth   1943,      75,76,77,      93,97,      00 (SEQ ID NO: 130)hsdnetqmaklygdskpqk    1943,      75,76,77,      93 (SEQ ID NO: 131)hfanlkgtqtrgk          1959 (SEQ ID NO: 132) hfanlkgtktrgk               1976,   89,90,         99,00,01 (SEQ ID NO: 114)hfanlkgtktrgklcpk                1976,      90             00,01(SEQ ID NO: 115) kprsalkckgflh                      1988(SEQ ID NO: 133) kctgtipsakasilhevk                            1993(SEQ ID NO: 121) hnvinaekapggpyk                           1993,97,      00 (SEQ ID NO: 126)hsdnetqmaklygdsk                            1993,97,      00(SEQ ID NO: 127) hsdneiqmvklygdskpqk                           1997,   98,   00 (SEQ ID NO: 118)kctgtipsakasilh                                        2000(SEQ ID NO: 120) kskpyytgehakai(g/a)ncpiwvk                                       2000 (SEQ ID NO: 134) 1.Influenza B has not been responsible for any human pandemic. 2.Abbreviation for years: e.g., “43” = 1943, “01” = 2001. 3. The firstyear that a given Replikin appears is indicated at the beginning of theseries of years in which that Replikin has been found. 4. OverlappingReplikin sequences are listed separately. 5. Return of replikins, absentfor several years, in the two years before the epidemic of 1977,underlined, correlates with increased total Replikin concentration(Replikin Count = number of Replikins per 100 amino acid residues). SeeFIG. 7.

TABLE 4HIN1 Replikin Sequences present in HINT hemagglutinins of Influenza viruses in each year for which aminoacid sequences were available (1918–2000) HIN1 Replikin Year Detected in Influenza HIN1 StrainPeak in FIG. 7: P1 E1 E1.1,1.2,1.3 E1.4 )hp(v/i)tigecpkyv(r/k)(s/t)(t/a)k1918,25,28,30,31,35,47,48,51,52,55,56,57,59,63,77,79,80,81,85,87,88,89,91,92,95,96,97,98,99,00(SEQ ID NO: 135) hdsnvknly(e/g)kv(k/r)(n/s)ql(k/r)nnak1918,   28,30,31,                              77,79,80,         88,   91,   95,      98(SEQ ID NO: 136) hdsnvknly(e/g)kv(k/r)(n/s)qlk1918    28,30,31,                              77,79,80,         88,   91,   95,      98(SEQ ID NO: 137) bkc(nn/dd)(a/t/e)cmesv(r/k)ngtydypkyseesklnre(e/k)1918,      30,   35,                           77,   80,                              98idgvk (SEQ ID NO: 138) hkc(nn/dd)(a/t/e)cmesv(r/k)ngtydypkyseesk1918,      30,   35,                           77,   80,                              98(SEQ ID NO: 139) hqn(e/g)qgsgyaadqkstqnai(d/n)gitnkvnsviekmntqftav1918,   28,30,31,35,                     59,      79,                        95gkefnklek (SEQ ID NO: 140) hqn(e/g)qgsgyaadqkstqnai(d/n)gitnkvnsviek1918,   28,30,31,35,                     59,      79,                        95(SEQ ID NO: 141) hqn(e/g)qgsgyaadqkstqnai(d/n)gitnk1918,   28,30,31,35,                     59,      79,                        95(SEQ ID NO: 142) kfeifpktsswpnh1918,                                          77 (SEQ ID NO: 143)kg(n/s/t)sypkl(n/s)ksy(v/t)nnkgkevlvlwgvh1918             35,                           77                               96(SEQ ID NO: 144) ksy(v/t)nnkgkevlvlwgvh1918             35,                           77                               96(SEQ ID NO: 145) hkcnnecmesvkngtydypkyseesklnrekidgvk      1928,   31,                                                            95(SEQ ID NO: 146) hkcnnecmesvkngtydypkyseesk      1928,   31,                                                            95(SEQ ID NO: 147) hkcnnecmesvkngtydypk      1928,   31,                                                            95(SEQ ID NO: 148) hkcnnecmesvk      1928,   31,                                                            95(SEQ ID NO: 149) hngkssfy(k/r)nllwlt(e/g)knglypnlsksyvnnkek      1928,                                                                  95,            00(SEQ ID NO: 150) hngkssfy(k/r)nllwlt(e/g)knglypnlsksyvnnk      1928,   31,                                                            95,            00(SEQ ID NO: 151) hngkssfy(k/r)nllwlt(e/g)knglypnlsk      1928,   31,                                                            95,            00(SEQ ID NO: 152) hngkssfy(k/r)nhlwlt(e/g)k      1928,   31,                                                            95,            00(SEQ ID NO: 153) kssfyknllwlteknglypnlsksyvnnkekevlvlwgvh      1928,   31,                                                            95(SEQ ID NO: 154) knllwlteknglypnlsksyvnnkekevlvlwgvh      1928,   31,                                                            95(SEQ ID NO: 155) knglypnlsksyvnnkekevlvlwgvh      1928,   31,                                                            95,96,         00(SEQ ID NO: 156) ksy(v/a)nnkekev(l/-)(v/-)lwgvh      1928,   31,         51,                                                95,96,   98,   00(SEQ ID NO: 157) kesswpnhtvtk      1928,   31,                                                            95(SEQ ID NO: 158) het(t/n)kgvtaacpyagassfyrnllwlvkkensypklsksyvnnk         1930,   35 (SEQ ID NO: 159)het(t/n)kgvtaacpyagassfyrnllwlvkkensypklsk          1930,   35(SEQ ID NO: 160) kfeifpktsswpnevlvlwgvh          1930 (SEQ ID NO: 161)kerswpkh                   1947,   51,52,55,56,            79,      82(SEQ ID NO: 162) klsksyvnnkekevlvlwqvh                   1947,   51(SEQ ID NO: 163) knnkekevlvlwqvh                   1947 (SEQ ID NO: 164)h(k/n)(g/q)kssfy(r/k)nllwltekng(l/s)yp(n/t)lsksya                     1948,                        79,               89,         96nnkek (SEQ ID NO: 165) h(k/n)(g/q)kssfy(r/k)nllwltek                     1948,                        79,               89,         96(SEQ ID NO: 166) hakkssfyk                         1951,         57,59(SEQ ID NO: 167) hngklcrlkgk                        1951,52,55,56,57,59,      79 (SEQ ID NO: 168)hyklnn(q/g)kk                                 1956,                                                      00(SEQ ID NO: 169) hdiyrdeainnrfqiqgvkltqgyk                                 1956 (SEQ ID NO: 170) kgngcfeifhk                                 1956 (SEQ ID NO: 171)klnrliektndkyhqiek                                  1956(SEQ ID NO: 172) klnrliektndkyh                                  1956(SEQ ID NO: 173) kchtdkgslsttk                                  1956(SEQ ID NO: 174) kinngdyaklyiwgvh                                  1956,(SEQ ID NO: 175) hngklcrkgiaplqlgk                                       1959,               82(SEQ ID NO: 176) hetnrqvtaacpyagansffrnliwlvkkessypklsk                                          1963,         81(SEQ ID NO: 177) hetnrqvtaacpyagansffrnliwlvkkessypk                                          1963,         81(SEQ ID NO: 178) hpptstdqqslyqnadayifvgsskynrkfk                                          1963,         81(SEQ ID NO: 179) hpptstdqqslyqnadayifvgsskynrkfkpeia                                          1963,         81(SEQ ID NO: 180) hdiyrdeainnrfqiqgvkitqgyk                                             1977,79,                  91(SEQ ID NO: 181) hqneqgsgyaadqkstqnaidgitnkvnsviekmntqftavgk                                             1977 (SEQ ID NO: 182)hqneqgsgyaadqkstqnaidgitnkvnsviek                                             1977 (SEQ ID NO: 183)hqneqgsgyaadqkstqnaingitnkvnsviekmntqftavgkefhkle                                                1979                   91k (SEQ ID NO: 184) hngklcrlkgiaplqlgk                                                1979 (SEQ ID NO: 185)hkcnnecmesvk                                                 1979(SEQ ID NO: 186) kfeifpkasswpnh                                                      1981(SEQ ID NO: 187) hdsnvknlyekvrsqlmnak                                                      1981(SEQ ID NO: 188) kvnsvikkmntqfaavgkefnh                                                      1981(SEQ ID NO: 189) khngklck                                                      1981(SEQ ID NO: 190) kkgtsypklsksythnkgkevlvlwgvh                                                      1981(SEQ ID NO: 191) kgtsypklsksythnkgkevlvlwgvh                                                      1981(SEQ ID NO: 192) klsksythnkgkevlvlwgvh                                                      1981(SEQ ID NO: 193) ksythnkgkevlvlwgvh                                                      1981(SEQ ID NO: 194) kgvtascshk                                                         1985,87(SEQ ID NO: 195) kgvtascshkgrssfyrnhlwlteknglypnlsk                                                         1985,87(SEQ ID NO: 196) kgnsypklsksyvnnkekevlvlwgih                                                               1988(SEQ ID NO: 197) kefnhlek                                                               1988(SEQ ID NO: 198) hpptstdqqslyqnadayvfvgsskynkkflkpeiatrpk                                                               1988(SEQ ID NO: 199) hpptstdqqslyqnadayvfvgsskynkkfk                                                               1988(SEQ ID NO: 200) hegkssfyrnllwltekegsypklknsyvnk                                                                     1991(SEQ ID NO: 201) hegkssfyrnllwltekegsypk                                                                     1991(SEQ ID NO: 202) hkcdnecmesvrngtydypkyseesk                                                                     1991(SEQ ID NO: 203) kesswpnhtvtk                                                                     1991,92(SEQ ID NO: 204) knllwlteknglypnlsksyvnnkekeilvlwgvh                                                                     1991,92,   96(SEQ ID NO: 205) hngkssfy(k/m)(n/-)llwlt(e/g)(-/k)knglypnlsk                                                                     1991,92,   96,         00(SEQ ID NO: 206) hngkssfyknhlwltek                                                                     1991,92,   96(SEQ ID NO: 207) htvtkgvtascshngkssfyknllwlteknglypnlsksyvnnkekevl                                                                           1995vlwgvh (SEQ ID NO: 208)htvt(k/g)gv(t/s)ascshngkssfy(k/m)(n/-)llwlt(e/g)k                                                                           1995,            00(-n/k)glypnlsk (SEQ ID NO: 209) htvtkgvtascshngkssfyknhlwltek                                                                           1995(SEQ ID NO: 210) kyvrstklrmvtglmipsiqsrglfgaiagfieggwtgmidgwygyh                                                                           1995(SEQ ID NO: 211) hqneqgsgyaadqkstqnaingitnkvnsiiekmntqftavgk                                                                           1995(SEQ ID NO: 212) hqneqgsgyaadqkstqnaingitnkvnsiiek                                                                           1995(SEQ ID NO: 213) hqneqgsgyaadqkstqnaingitnk                                                                           1995(SEQ ID NO: 214) hsgarsfyrnllwivkkgnsypk                                                                              1996(SEQ ID NO: 215) hsgarsfyrnllwivkkgnsypklnk                                                                              1996(SEQ ID NO: 216) hsgarsfyrnllwivkkgnsypklnksytndk                                                                              1996(SEQ ID NO: 217) hsgarsfyrnllwivkkgnsypklnksytndkgk                                                                              1996(SEQ ID NO: 218) htvskgvttscshngk                                                                              1996(SEQ ID NO: 219) katswpnhettk                                                                              1996(SEQ ID NO: 220) kqvttscshnqk                                                                              1996(SEQ ID NO: 221) kgnsypklnksytndkgkevlviwgvh                                                                              1996(SEQ ID NO: 222) klnksytndkgkevlviwgvh                                                                              1996(SEQ ID NO: 223) ksytndkgkevlviwgvh                                                                              1996(SEQ ID NO: 224) hnqkssfyrnllwlt(e/q)knglypnlsksy(v/a)annkek                                                                                 1997,98,99(SEQ ID NO: 225) hpitigecpkyvrsak                                                                                 1997(SEQ ID NO: 226) hqneqgsgyaadqkstqnaingimkvnsviekmntqftavgk                                                                                    1998(SEQ ID NO: 227) hqneqgsgyaadqkstqnaingitnkvnsviek                                                                                    1998(SEQ ID NO: 228) hngkssfyrnllwlteknglypnlsksyvnnkek                                                                                    1998(SEQ ID NO: 229) 1. Influenza HIN1 was responsible for the humanpandemic (global distribution) of 1918. 2. Abbreviation for years: eg.“96” = 1996. 3. The first year that a given Replikin appears isindicated at the beginning of the series of years in which that Replikinhas been found in this work. 4. Overlapping Replikin sequences arelisted separately. 5. Increase in number of new Replikin structuresoccurs in years of epidemics (underlined): eg. 1918 and 1977 andcorrelates with increased total Replikin concentration (number ofReplikins per 100 amino acid residues). See FIG. 7.

TABLE 5Replikin Sequences present in hemagglutinins of Influenza H2N2 viruses in years1957–2000 Influenza H2N2 Replikins Year Detected in Influenza H2N2 strain(Peak in FIG. 8: P2 E2 ) khfekvkilpk1957,58,59,60,61,64,6568,         78,83,84,91 (SEQ ID NO: 230)khllssvkhfekvk 1957,58,59,60,61,                     83,84,91(SEQ ID NO: 231) ha(k/q/m)(d/n)ilekthngk1957,58,59,60,61,64,6568,         78,83,84,91,   95 (SEQ ID NO: 232)ba(k/q/m)(d/n)ilekthngklc(k/r)1957,58,59,60,61,64,6568,         78,83,84,91,   95 (SEQ ID NO: 233)hnvhpltigecpkyvksek 1957,58,59,         65,68 (SEQ ID NO: 234)hpltigecpkyvksek 1957,58,59,         65,68,64,6568,78,83,84,91(SEQ ID NO: 235) khllssvkhfekvkilpk 1957,58,59,60,61,64,6568,         78(SEQ ID NO: 236) krqssgimktegtlencetkcqtplgainttlpfhnvh1957,   59,                           83 (SEQ ID NO: 237)kgsnyp(v/i)ak(g/r)synntsgeqmliiwq(v/i)h1957,58,59,   61,                     83,   91,   95 (SEQ ID NO: 238)httlgqsracavsgnpsffrnmvwltekgsnypvak 1957 (SEQ ID NO: 239) klifekvk1957,   59,         65 (SEQ ID NO: 240)kiskrgssgimktegtlencetkcqtplgainttlpfh1957,   59,         65,                     91 (SEQ ID NO: 241)krgssgimktegtlencetkcqtplgainttlpfh1957,   59,         65,                     91 (SEQ ID NO: 242)ktegtlencetkcqtplgainttlpfh1957,   59,         65,                     91 (SEQ ID NO: 243)kiskrgssgimktegtlencetkcqtplgainttlpfli1957,   59,         65,                     91 (SEQ ID NO: 244)ktegtlencetkcqtplgainttlpfhn(v/i)h1957,   59,         65,                     91 (SEQ ID NO: 245)kiskrgssgimktegtlencetkcqtplgainttlpfh1957,   59,         65,                     91 (SEQ ID NO: 246)k(e/g)snypvakgsynntsgeqmliiwgvh 1957,      60,      65 (SEQ ID NO: 247)hpltigecpkyvksek 1957,      60,      65 (SEQ ID NO: 248)kcqtplgaikttlpfh 1957,               65 (SEQ ID NO: 249)hhsndqgsgyaadkestqka(f/i)dgitnkvnsviek-            1961,   65,68,            83,84 mntqfeavgklf(n/s)nleklenlnkk(SEQ ID NO: 250) hsndqgsgyaadkestqka(f/i)dgitnkvnsviek-            1961,   65,68,            83,84 mntqfeavgklf(n/s)nleklenlnkk(SEQ ID NO: 251) hsndqgsgyaadkestqka(f/i)dgitnk            1961,   65,68,            83,84 (SEQ ID NO: 252)hdsnvmlydkvrmqlrdnak                1964,   68,76,            84,91(SEQ ID NO: 253) hkcddecmnsvkngtydypklnmeikgvk               1964,6568,76,         83,84,91 (SEQ ID NO: 254)hkcddecmnsvkngtydypklnrneik               1964,6568,76,         83,84,91 (SEQ ID NO: 255)hkcddecmnsvkngtydypk                1964,6568,76,         83,84,91(SEQ ID NO: 256) hkcddecmnsvk               1964,6568,76,         83,84,91 (SEQ ID NO: 257)kgsnypvakgsynntngeqiliiwgvh                         1976,78(SEQ ID NO: 258) hsndqgsgyaadkestqkavdgitnkvnsviekmntqfeavgk                        1976,               91 (SEQ ID NO: 259)krgssgimktegtlencetkcqtplgainttlpfh                        1976,78,      83,84 (SEQ ID NO: 260)hpltigecpkyvksek                         1976 (SEQ ID NO: 261)hakdilekthngklck                         1976 (SEQ ID NO: 262) 1.Influenza H2N2 was responsible for the human pandemic (globaldistribution) of 1957. 2. Abbreviation for years: eg. “58” = 1958. 3.The first year that a given Replikin appears is indicated at thebeginning of the series of years in which that Replikin has been foundin this work. 4. Overlapping Replikin sequences are listed separately.5. Increase in number of new Replikin structures occurs in years ofepidemics (underlined): eg. 1957 and 1965 and correlates with increasedtotal Replikin concentration (number of Replikins per 100 amino acidresidues). See FIG. 8.

TABLE 6H3N2 Replikin Sequences present in H3N2 hemagglutinins of Influenza virusesin each year for which amino acid sequences were available (1968–2000)Influenza H3N2 Renlikins Year Detected in Influenza H3N2 strain Influenza Replikins(Peak in FIG. 8: P3 E3 E4) hdvyrdealnnrfqikgvelksgyk1968,72,  75,                                                       96,97,98(SEQ ID NO: 263) htidltdsemnklfertrk 1968 (SEQ ID NO: 264) kfhqiek1968,72,  75,   77                                                  96,97,98(SEQ ID NO: 265) ktnekfh(g/q)iek1968                                     86                               98(SEQ ID NO: 266) klnr(v/l)iektnekfli1968,72,  75,   77                                                     97,98(SEQ ID NO: 267) hqiekefsevegriqdlekyvedtk1968,72,                                                                  98(SEQ ID NO: 268) kicnnphk         1975 (SEQ ID NO: 269) klnrvikktnekfh        1975 (SEQ ID NO: 270) hd(i,v)yrdealnnrfqik(g/q)ve(r/k)s(q/g)yk         1975,76,77,                     86 (SEQ ID NO: 271)hqiekefsevegriqdlekyvedtk          1975 (SEQ ID NO: 272)kyvedtkidlwsynaellvalenqh          1975 (SEQ ID NO: 273)kyvkqnslklatgmrnvpekqtrglfgaiagfiengwegmidgwygfrh          1975(SEQ ID NO: 274) kefsevegriqdlekyvedtkidlwsynaellvalenqh         1975                                                              2000(SEQ ID NO: 275) hqn(sfe)(e/q)g(tIs)g(q/y)aad(l/q)k-         1975                                                              2000-stq(a/n)a(i/l)d(q/g)I(n/t)(g/n)k(l/v)n (r/s)vi(e/c)k (SEQ ID NO: 276)hcd(g/q)f(q,r)nekwdlf(v,/i)er(s/t)k         1975,76,77,78,80,81,82,83,84,85,86,88,89,90,91,92,93,94,95,96,97,98(SEQ ID NO: 277) htidltdsemnkklfertrk                1977,(SEQ ID NO: 278) ksgstypvlkvtmpnndnfdklyiwgvh                1977(SEQ ID NO: 279) klnwltksgntypvlnvtmpnndnfdklviwgvh                           1982 (SEQ ID NO: 280) htidltdsemnklfektrk                                       1986 (SEQ ID NO: 281)klnrliektnekfhqtek                                          1987(SEQ ID NO: 282) htgkssvmrsdapidfcnsecitpnqsipndkpfqnvnkitygacpk                                                            1994(SEQ ID NO: 283) htgkssvmrsdapidfcnsecitpnqsipndkpfqnvnk                                                            1994(SEQ ID NO: 284) hpstdsdqtslyvrasgrvtvstkrsqqtvipk                                                            1994(SEQ ID NO: 285) kyvedtkidlwsynaellvalenqh                                                                     1997,98(SEQ ID NO: 286) klfertrkqlrenaedmgngcfldyh                                                                        1998(SEQ ID NO: 287) krrsiksffsrlnwlh                                                                        1998(SEQ ID NO: 288) hpvtigecpky(v/r)kstk                                                                           2000(SEQ ID NO: 289) kgnsypklsklsksyiinkkkevlviwgih                                                                           2000(SEQ ID NO: 290) klsklsks(v/y)iinkkkevlviwgih                                                                           2000(SEQ ID NO: 291) klsks(v/y)iinkkkevlviwgih                                                                           2000(SEQ ID NO: 292) 1. Influenza H3N2 was responsible for the humanpandemic (global distribution) of 1968. 2. Abbreviation for years: eg.“77” = 1977. 3. The first year that a given Replikin appears isindicated at the beginning of the series of years in which that Replikinhas been found. 4. Overlapping Replikin sequences are listed separately.5. Increase in number of new Replikin structures occurs in years ofepidemics (underlined) : eg. 1975 and correlates with increased totalReplikin concentration (number of Replikins per 100 amino acidresidues). See FIG. 8.

Several properties of Replikin concentration are seen in FIG. 7 and FIG.8 to be common to all four influenza virus strains. First, theconcentration is cyclic over the years, with a single cycle of rise andfall occurring over a period of two to thirty years. This rise and fallis consistent with the known waxing and waning of individual influenzavirus strain predominance by hemagglutinin and neuraminidaseclassification. Second, peak Replikin concentrations of each influenzavirus strain previously shown to be responsible for a pandemic wereobserved to relate specifically and individually to each of the threeyears of the pandemics. For example, for the pandemic of 1918, where theinfluenza virus strain, H1N1, was shown to be responsible, a peakconcentration of the Replikins in H1N1 independently occurred (P1); forthe pandemic of 1957, where H2N2 emerged and was shown to beresponsible, a peak concentration of the Replikins in H2N2 occurred(P2); and for the pandemic of 1968, where H3N2 emerged and was shown tobe the cause of the pandemic, a peak concentration of the Replikins inH3N2 occurred (P3). Third, in the years immediately following each ofthe above three pandemics, the specific Replikin concentration decreasedmarkedly, perhaps reflecting the broadly distributed immunity generatedin each case. Thus, this post-pandemic decline is specific for H1N1immediately following the pandemic (P1) for which it was responsible,and is not a general property of all strains at the time. An increase ofReplikin concentration in influenza B repeatedly occurred simultaneouslywith the decrease in Replikin concentration in H1N1, e.g., EB1 in 1951and EB2 in 1976, both associated with influenza B epidemics having thehighest mortality. (Stuart-Harris, et al., Edward Arnold Ltd. (1985).Fourth, a secondary peak concentration, which exceeded the primary peakincrease in concentration, occurred 15 years after each of the threepandemics, and this secondary peak was accompanied by an epidemic: 15years after the 1918 pandemic in an H1N1 ‘epidemic’ year (E1); eightyears after the 1957 pandemic in an H2N2 ‘epidemic’ year (E2); andoccurred seven years after the 1968 pandemic in an H3N2 ‘epidemic’ year(E3). These secondary peak concentrations of specific Replikins mayreflect recovery of the strain. Fifth, peaks of each strain's specificReplikin concentration frequently appear to be associated with declinesin Replikin concentration of one or both other strains, suggestingcompetition between strains for host sites. Sixth, there is an apparentoverall tendency for the Replikin concentration of each strain todecline over a period of 35 years (H2N2) to 60 years (influenza B). Thisdecline cannot be ascribed to the influence of vaccines because it wasevident in the case of influenza B from 1940 to 1964, prior to commonuse of influenza vaccines. In the case of influenza B, Replikin recoveryfrom the decline is seen to occur after 1965, but Replikin concentrationdeclined again between 1997 and 2000 (FIG. 7). This correlates with thelow occurrence of influenza B in recent case isolates. H1N1 Replikinconcentration peaked in 1978-1979 (FIG. 7) together with thereappearance and prevalence of the H1N1 strain, and then peaked in 1996coincident with an H1N1 epidemic. (FIG. 7). H1N1 Replikin concentrationalso declined between 1997 and 2000, and the presence of H1N1 strainsdecreased in isolates obtained during these years. For H2N2 Replikins,recovery from a 35 year decline has not occurred (FIG. 8), and thiscorrelates with the absence of H2N2 from recent isolates. For H3N2, theReplikin concentration of many isolates fell to zero during the periodfrom 1996 to 2000, but other H3N2 isolates showed a significant, sharpincrease in Replikin concentration. This indicates the emergence of asubstrain of H3N2, which is designated herein as H3N2(R).

FIGS. 7 and 8 demonstrate that frequently, a one to three year stepwiseincrease is observed before Replikin concentration reaches a peak. Thisstepwise increase proceeds the occurrence of an epidemic, which occursconcurrently with the Replikin peak. Thus, the stepwise increase inconcentration of a particular strain is a signal that particular strainis the most likely candidate to cause an epidemic or pandemic.

Currently, Replikin concentration in the H3N2(R) strain of influenzavirus is increasing (FIG. 8, 1997 to 2000). Three similar previous peakincreases in H3N2 Replikin concentration are seen to have occurred inthe H3N2-based pandemic of 1968 (FIG. 8), when the strain first emerged,and in the H3N2-based epidemics of 1972 and 1975 (FIG. 8). Each of thesepandemic and epidemics was associated with excess mortality. (Ailing, etal., Am J. Epidemiol., 113(1):30-43 (1981). The rapid ascent inconcentration of the H3N2(R) subspecies of the H3N2 Replikins in1997-2000, therefore, statistically represents an early warning of anapproaching severe epidemic or pandemic. An H3N2 epidemic occurred inRussia in 2000 (FIG. 8, E4); and the CDC report of December 2001 statesthat currently, H3N2 is the most frequently isolated strain of influenzavirus worldwide. (Morbidity and Mortality Weekly Reports (MMWR), Centerfor Disease Control; 50(48): 1084-68 (Dec. 7, 2001).

In each case of influenza virus pandemic or epidemic new Replikinsemerge. There has been no observation of two of the same Replikins in agiven hemagglutinin in a given isolate. To what degree the emergence ofa new Replikin represents mutations versus transfer from another animalor avian pool is unknown. In some cases, each year one or more of theoriginal Replikin structures is conserved, while at the same time, newReplikins emerge. For example, in influenza virus B hemagglutinin, fiveReplikins were constantly conserved between 1919 and 2001, whereas 26Replikins came and went during the same period (some recurred afterseveral years absence). The disappearance and re-emergence years laterof a particular Replikin structure suggests that the Replikins returnfrom another virus host pool rather than through de novo mutation.

In the case of H1N1 Replikins, the two Replikins present in the P1 peakassociated with the 1918 pandemic were not present in the recovery E1peak of 1933, which contains 12 new Replikins. Constantly conservedReplikins, therefore, are the best choice for vaccines, either alone orin combination. However, even recently appearing Replikins accompanyingone year's increase in concentration frequently persist and increasefurther for an additional one or more years, culminating in aconcentration peak and an epidemic, thus providing both an early warningand time to vaccinate with synthetic Replikins (see for example, H1N1 inthe early 1990's, FIG. 7; see also, for example, H5N1 1995-2002, FIG.11, “Replikin Count” (number of Replikins per 100 amino acids) refers toReplikin concentration) and FIG. 15).

The data in FIGS. 7, 8, 11 and 15 demonstrate a direct relationshipbetween the presence and concentration of a particular Replikin ininfluenza protein sequences and the occurrence of pandemics andepidemics of influenza. Thus, analysis of the influenza virushemagglutinin protein sequence for the presence and concentration ofReplikins provides a predictor of influenza pandemics and/or epidemics,as well as a target for influenza vaccine formulation. It is worthnoting again with reference to this data, previously, no strain-specificchemical structures were known with which to predict the strains thatwould predominate in coming influenza seasons, nor to devise annualmixtures of whole-virus strains for vaccines.

Similar to the findings of strain-specific Replikin Count increases inthe influenza group one to three years prior to the occurrence of astrain-specific epidemics, the increase in Replikin Count of thecoronavirus nucleocapsid protein has also been identified. ReplikinCounts of the coronavirus nucleocapsid protein has increased as follows:3.1 (±1.8) in 1999; 3.9(±1.2) in 2000; 3.9 (±1.3) in 2001; and 5.1(±3.6) in 2002. This pre-pandemic increase supports the finding that acoronavirus is responsible for the current (2003) SARS pandemic. (SeeTable 7)

Thus, monitoring Replikin structure and Replikin Count provides a meansfor developing synthetic strain-specific preventive vaccination andantibody therapies against the 1917-1918 Goose Replikin and its modifiedand accompanying Replikins as observed in both influenza and coronavirusstrains.

FIG. 10 depicts the automated Replikin analysis of nucleocapsidcoronavirus proteins for which the protein sequence is available onisolates collected from 1962 to 2003. Each individual protein isrepresented by an accession number and is analyzed for the presence ofReplikins. The Replikin Count (number of Replikins per 100 amino acid)is automatically calculated as part of the automated Replikin analysis.For each year, the mean (±Standard deviation (S.D.)) Replikin Count peryear is automatically calculated for all Replikin Counts that year. Thisexample of early warning of increasing replication, before an epidemic,of a particular protein (the nucleocapsid protein) in a particular virusstrain (the coronavirus) is comparable to the increase seen in strainsof influenza virus preceding influenza epidemics and pandemics (FIGS. 7,8, 11 and 15). It may be seen that the Replikin Count rose from 1999 to2002, consistent with the SARS coronavirus pandemic, which emerged atthe end of 2002 and has persisted into 2003. FIG. 9 provides a graph ofthe Replikin Counts for several virus strains, including the coronavirusnucleocapsid Replikin, from 1917 to 2002.

TABLE 7 ‘Multi-K’ % Replikins:  Untreated Replikin Sequence LengthMortality ORGANISM                  1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 21 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9Amino Acid position A. INFLUENZA, SARS AND OTHER CORONAVIRUSESk k g t s y p k l s k s y t n n k g k e v l v l w g v h h  291917–18 Goose Replikin (SEQ ID NO: 17)k k g t s y p k l s k s y t n n k g k e v l v l w g v h h  29  251918 Human Influenza (SEQ ID NO: 293)l k e d l y p k l r k s v v h n k k k e v l v i w g i h h  291919–2001 HIN1, HIN2 (SEQ ID NO: 294)l k e n s y p k l r k s i i i n k k k e v l v i w g i h h H3N2 Influenza(SEQ ID NO: 295)k k g t s y p k l s k s y t n n k k k e v l v l w g v h h  292001 HIN2 Influenza (SEQ ID NO: 296)k k n s a y p t l k r s y n n t n q e d l l v l w g i h h >371996–2001 H5N1 Influenza (SEQ ID NO: 297)k k s a k t g t p k p s r n q s p a s s q t s a k s l a h >372000 Human cornoavirus 229E (SEQ ID NO: 298)¹k k l g v d t e k q q q r s k s k e r s n s k t r d t t p >372003 Cancine corona- virus (SEQ ID NO: 299)²k n g l y p n l s k s y a n n k e k e v l i l w g v h h  28 2002 HIN2(SEQ ID NO: 300) k k i n s p q p k f e g s g v p d n e n l k t s q q h 27 Avian bronchitis corona- virus (SEQ ID NO: 301)k t g n a k l q r k k e k k n k r e t t l q q h  24 Porcine epidemicdiarrhea coronavirus (SEQ ID NO: 302)k h l d a y k t f p p t e p k k d k k k k  21 2003 Human SARS nucleo-capsid (SEQ ID NO: 303) k h r e f v f k n k d g f l y v y k  192003 Human SARS spike protein (SEQ ID NO: 304) k e e l d k y f k n h  112003 Human SARS spike protein (SEQ ID NO: 305) k y r y l r h g k   92003 Human SARS spike protein (SEQ ID NO: 306) k k g a k l l h k   9  552003 SARS envelope protein (SEQ ID NO: 307) k h l d a y k   7  552003 Human SARS nucleo- capsid protein (SEQ ID NO: 308)B. OTHER VIRUSES, BACTERIA, MALARIA AND CANCER REPLIKINSh   l v c   g k k g l g l s g r k k  19 HIV-TAT (SEQ ID NO: 309)k k i t n i t t k f e q l e k c c k h  19 Monkeypox virus(SEQ ID NO: 310) k k l k k s l k l l s f y h p k k  17African swine fever virus (SEQ ID NO: 311)k n r i e r l k k e y s s t w h  16 West Nile Virus (SEQ ID NO: 312)k s r g i p i k k g h  11 Nipah virus, v-protein (SEQ ID NO: 313)k s r i m p i k k g h  11 Hendra virus, V-protein (SEQ ID NO: 314)k k f l n q f k h h  10 Sindbis virus (SEQ ID NO: 315)k k k s k k h k d k  10 EEL Leukemia (SEQ ID NO: 85) k h h p k d n l i k 10 BRCA-1 Breast cancer (SEQ ID NO: 81) k h k r k k f r q k  10Ovarian cancer (SEQ ID NO: 84) k a g v a f l h k k  10 >90%Glioma Replikin (SEQ ID NO: 83) k i h l i s v k k   9 Smallpox virus(SEQ ID NO: 316) k l i s i h e k   8 Smallpox virus (SEQ ID NO: 317)k l r e e h e k   8 B. anthracis, HATPase (SEQ ID NO: 318)k h k k q i v k   8 Plasm Falciparum ATPase (SEQ ID NO: 319)k k h a t v l k   8 >90% Ebola virus poly- merase (SEQ ID NO: 320)k k e d d e k h   8 P. falciparum blood trophozoites (SEQ ID NO: 321)k h k e k m s k   8 >90% (K-RAS 2B) lung cancer (SEQ ID NO: 322)k k l r h e k   7 Rous sarcoma virus (SEQ ID NO: 48) k k l r h e k   7c-src, colon, breast cancer (SEQ ID NO: 52) k k l r h d k   7c-yes, melanoma, colon cancer (SEQ ID NO: 50) ¹Human coronavirus 229E2000, SEQ ID NO: 532: kksaktgtpkpsrnqspassqtsakslarsqssetkeqkh ²Caninecoronavirus 2003, SEQ ID NO: 533: kklgvdtekqqqrsrskskersnsktrdttpknenkhSARS and H3N2-Fujian Influenza Virus Replikins Traced Back to a 1918Pandemic Replikin

The origin of the SARS virus is as yet unknown. We report evidence thatcertain SARS virus peptides can be traced back through homologouspeptides in several strains of influenza virus isolates from 2002 to asequence in the strain of the 1918 influenza pandemic responsible forthe deaths of over 20 million people.

By quantitative analysis of primary protein sequences of influenza virusand other microorganisms recorded through the last century we have founda new class of peptide structures rich in lysines and histidine, relatedto the phenomenon of rapid replication itself and to epidemics, ratherthan to the type of organism (e.g. Table 1) and named them Replikins. Wehave found a new class of peptide structures with the followingobligatory algorithm: at least two lysines 6 to 10 residues apart,lysine concentration 6% or greater, one histidine, in 7 to 50 aminoacids. Because these peptides relate to the phenomenon of rapidreplication itself and to epidemics, we named them Replikins. We havefound a quantitative correlation of strain-specific replikinconcentration (replikin count=number of replikins per 100 amino acids)in the hemagglutinin protein with influenza epidemics and pandemics(FIG. 7). No previous correlation of influenza epidemics withstrain-specific viral protein chemistry has been reported. Conservation,condensation and concentration of replikin structure also has been foundin influenza (e.g. in Table 7a), HIV and malaria. The detection ofreplikins in SARS coronavirus, in addition to tracing its possibleevolution, has permitted the synthesis of small SARS antigens forvaccines.

We have found a quantitative correlation of strain-specific replikinconcentration (count) in the influenza hemagglutinin proteins withinfluenza epidemics and with each of the three pandemics of the lastcentury, in 1918, 1957, and 1968. A similar course was observed for eachof these three pandemics: after a strain-specific high replikin count,an immediate decline followed, then a ‘rebound’ increase with anaccompanying epidemic occurred. Also, a 1 to 3 year warning increase incount preceded most epidemics.

We found that the replikin in the hemagglutinin of an influenza virusisolated from a goose in 1917 (which we named the Goose Replikin)appeared in the next year in the H1N1 strain of influenza responsiblefor the 1918 pandemic, with only two substitutions as follows:kkg(t/s)sypklsksy(t/v)nnkgkevlvlwgvhh (SEQ ID NO: 323). Table 7a showsthat the influenza 1917 Goose Replikin (GR) then was essentiallyconserved for 85 years, despite multiple minor substitutions andapparent translocations to other influenza strains. We have found thatthe 1917 influenza GR demonstrated apparent mobility between severalinfluenza strains, appearing in H1N1 (the pandemic of 1918), in H2N2(pandemic of 1957-58), in H3N2 (pandemic of 1968, epidemic in China andRussia 2000, Fujian strain epidemic 2003) and in H5N1 (epidemic in China1997). In 1997 its structure was restored in H1N2 exactly to its 1918structure KKGSSYPKLSKSYVNNKGKEVLVLWGVHH (SEQ ID NO: 324).

The SARS coronavirus first appeared in the 2002-2003 influenza season.The dual origin in 2002 of SARS replikins, from influenza GR andcoronavirus replikins (or from some unknown shared precursor) issuggested by the following events, all of which occurred in 2002: 1) acondensation for the first time in 85 years is seen in the GR-H1N2Replikin sequence from 29 to 28 amino acids (Table 7a) (A similarcondensation was found in H3N2 Fujian from 29 to 27 amino acids in thecurrent epidemic (Table 7a)); 2) the replikin count of GR-H1N2 showed amarked decline consistent with GR moving out of H1N2; 3) the replikincount of coronavirus nucleocapsid proteins showed a marked increase; and4) SARS coronavirus appeared in 2002-2003 with replikins containing thefollowing motifs: ‘kkg’ and ‘k-k’, previously seen in GR 1918 andGR-H1N2 2001; ‘k-kk’, ‘kk’ and ‘kl’ seen in influenza GR-H1N2 2001; ‘kk’seen in the avian bronchitis coronavirus replikin; and ‘kk-kk-k’ (SEQ IDNO: 325), ‘k-k’, ‘kl’ and ‘kt’ seen in the replikin of porcine epidemicdiarrhea coronavirus (Table 7a) (SARS is believed to have made its firstappearance in humans as the epidemic pneumonia which erupted in acrowded apartment house where there was a severe back-up of fecalsewage, which was then airborne by ventilating fans).

TABLE 7aGoose Replikin (GR) sequences in different influenza strains from 1917 to2003; SARS and H3N2-Fujian appearance 2002-2003. ReplikinVirus or other organism Replikins related to the Goose Replikin: Lengthcontaining replikin Continuous amino acid sequences (Number(Complete replikins Shared motif and/or position not underlined of aminoexcept for Fujian strain) Amino acid substitutions underlined acid)‘Condensed’ indicates condensation ofsequence length in H1N2 and H3N2-Fujian SEQ. ID NO.kkgtsypklsksytnnkgkevlvlwgvhh 326 291917 H1N_ Influenza Goose Replikin (GR) kkgssypklsksyvnnkgkevlvlwgvhh327 29 1918 GR in H1N1 Human Influenza kkgsnypva k gsynntsgeqmliiwgvhh328 29 1958 GR H2N2 Influenza kkgpnypva k gsynntsgeqmliiwgvhh 329 291964, 1965, 1968 GR in H2N2 Influenza kkgtsypklsksytnnkgkevlvlwgvhh 33029 1976, '77, '80, '81, '85 GR in H1N1 Influenza kk nsaypti krsynntnqedllvlwgi hh 331 29 1996-2001 GR in H5N1 Influenzakkgdsypklsksytnnkgkevlviwgvhh 332 29 1996 GR in H1N1 Influenzakkgssypklsksyvnnkgkevlvlwgvhh 333 29 1997, 1998 GR in H1N1 Influenzakkgnsypk isksyinnk e kevlvlwgi hh 334 29 1999 GR in H1N2 Influenzakkgnsypklsksyinnk k kevlviwgi hh 335 29 2000 GR in H1N2 Influenzakkgnsypklsksyinnkgkkvlvlwgi hh 336 29 2001 GR in H1N2 Influenzakkgtsypklsksytnnk k kevlvlwgvhh 337 29 2001 GR in H1N2 Influenza knglypnlsksyannk e kevlilwgvhh 338 282002 GR in H1N2 Influenza (condensed) kh lday k tfpptep kk d kkkk 339 212002-3 Human SARS nucleocapsid protein kk ensypklr ksiiink k kevlviwgihh 340 29 1968-2001 GR in H3N2 Influenza (complete) k ley k ypalnvtmpnndk fd klyiwgvhh 341 29 1996 H3N2 Fujian Influenza (incomplete) k y kypalnvtmpnne k fd klyiwgvhh 342 272003 H3N2 Fujian (condensed, incomplete) k tgna k lqr kk e kknk rettlqqh 343 24 Porcine epidemic diarrhea coronavirus kk inspgpk fegsgvpdnenl ktsqq h 344 27 Avian bronchitis coronavirus kk nv k sa k qlp h l kvlldvrga k qlp h 345 27 2000 shrimp white spot syndrome virus kk inspgpkfegsgvpdnenl k tsqq h 346 27 Avian bronchitis coronavirus kh lrefvf k nk dgflyvy kk 347 20 2002-3 Human SARS spike protein kkga kll h k pivw h348 14 2002-3 Human SARS nucleocapsid protein kh lrefvf knk dgflyvy kk349 20 2002-3 Human SARS spike protein kkgakll hk pivw h 350 142002-3 Human SARS nucleocapsid protein k eeld k yf k n h 351 112002-3 Human SARS spike protein kkga kll hk 352 92002-3 Human SARS envelope protein k yrylr hgk 353 92002-3 Human SARS spike protein kh lday k 354 72002-3 Human SARS nucleocapsid protein ksrgipi kkgh 355 11Nipah virus, v-protein ksrimpi kkgh 356 11 Hendra virus, v-protein kkflnqf khh 357 10 Sindbis virus kkkskkhk d k 358 10 EEL leukemia khh p kdnli k 359 10 BRCA-1 breast cancer kh k r kk frqk 360 10 Ovarian cancerk agvafl hkk 361 10 Glioma Replikin k i h lisv kk 362 9 Smallpox virus krfil h a kk 363 9 HIV TAT protein klisi h e k 364 8 Smallpox virus klreeh c k 365 8 B. anthracis, HATPase kkh atvl k 366 8Ebola virus polymerase

The recent increasingly high replikin count peaks, including thepresence of the 1917 Goose Replikin (FIG. 7), now in HIN2 (Table 7a),approaching the 1917 replikin count, could be a warning of a comingpandemic which may already have begun since the SARS virus and theH3N2-Fujian virus are the current carriers of the short replikinderivatives of the Goose Replikin seen in Table 7 and 7a to beassociated with high mortality.

Since the Goose Replikin has at least an 85 year history involving mostor all of the A-strains of influenza and SARS, it and its components areconserved vaccine candidates for pan-strain protection. Condensed shortSARS replikins, 7 to 21 amino acids long, enriched in % lysine andhistidine compared to the Goose Replikin, occurred in association withthe higher mortality rate of SARS (10-55%) when compared to that (2.5%)of the Goose Replikin, 29 amino acids long. Short replikins here mixedwith long replikins in SARS may be responsible for high mortality. Thisis also the case for replikins of other organisms such as the ebola andsmallpox viruses and anthrax bacteria (Table 7a). These short SARSreplikins showed surprising homology with short replikins of otherorganisms such as smallpox, anthrax, and ebola which are associated witheven higher untreated mortality rates (Table 7a).

Short synthetic vaccines, besides being much more rapidly produced (daysrather than months), and far less expensive, should avoid the sideeffects attendant on the contamination and the immunologicalinterference engendered by multiple epitopes of thousands of undesiredproteins in current whole virus vaccines in general. In any case forinfluenza, current whole virus vaccines are ineffective in more thanhalf of the elderly. But would short replikins be sufficientlyimmunogenic? The short glioma replikin ‘kagvaflhkk’ (SEQ ID NO: 1)proved to be a successful basis for a synthetic anti-glioblastomamultiforme and anti-bronchogenic carcinoma vaccine. It producedanti-malignin antibody, which is cytotoxic to cancer cells atpicograms/cell and relates quantitatively to the survival of cancerpatients. In order to prepare for a recurrent SARS attack, which appearslikely because of the surge we found in the coronavirus nucleocapsidreplikin count in 2002, we synthesized four SARS short replikins, foundin nucleocapsid, spike, and envelope proteins. We found that thesesynthetic short SARS replikins when injected into rabbits also producedabundant specific antibody. For example, the 21 amino acid SARSnucleocapsid replikin antibody binds at dilutions greater than 1 in204,800. Because of previous unsuccessful attempts by others to achievewith various small peptides a strong immune response without theunwanted side effects obtained with a whole protein or the thousands ofproteins or nucleic acids as in smallpox vaccine, the ability of smallsynthetic replikin antigens to achieve strong immune responses issignificant for the efficacy of these SARS vaccines.

We examined the relationship of Replikin structure in influenza and SARSviruses to increased mortality, with results as shown in Table 7. Therelation of high mortality to short or condensed Replikin sequences isseen in the high mortality organisms shown in Section B of Table 7, inviruses other than influenza and SARS, and in bacteria, malaria andcancer. In support of the unifying concept of Replikin structure and ofthe relation of Replikins to rapid replication rather than any cell typeor infectious organism, in addition to the prevalence of the basicReplikin structure in a broad range of viral, bacterial, malarial andcancer organisms in which replication is crucial to propagation andvirulence, the following homologous sequences have been observed: notethe “k”s in positions 1 and 2, note the alignment of “k”s as they wouldpresent to DNA, RNA or other receptor or ligand for incorporation or tostimulate rapid replication, note the frequency of “double k”s and“multiple k”s, note the frequency of “g” in position 3 and theoccurrence of the triplets “kkg”, “hek”, “hdk” and “hkk” in the mostcondensed shortened Replikins associated with the highest mortalityorganisms, cancer cells and genes as diverse as the smallpox virus, theanthrax virus, Rous sarcoma virus and glioblastome multiforme (glioma),c-src in colon and breast cancer, and c-yes in melanoma and coloncancer. Note also the almost identical Replikin structure for tworecently emerging high mortality viruses in Australia and SoutheastAsia, Nipah and Hendrah viruses. These two viruses are reported to havesimilar or identical antibodies formed against them but no structuralbasis has been known for this up till now, with our finding of their twoalmost identical Replikins, for this similar antibody.

Table 7 also shows the relationship of five SARS Replikins of 2003 whichwe have found both to the influenza Goose Replikin of 1917 and to twocoronaviruses, the avian bronchitis coronavirus and the porcine epidemicdiarrhea virus. The first 2003 human SARS Replikin in Table 7 showscertain sequence homologies to the influenza virus goose 1917 and human1918 Replikins through an intermediary structure of influenza H1N2 in2002 (e.g., see Replikin “k” in positions 1, 18 and 19). The 1917 GooseReplikin sequence is seen in Table 7 to have been largely conserveddespite many substitutions in amino acids which are not crucial to thedefinition of Replikins through 1999 (substitutions are show initalics). The original 29 amino acid 1917 Replikin sequence was thenfound to have been almost exactly restored to its structure of 1917-1918in the 2001H1N2 Replikin. However, the 2002H1N2 influenza Replikin hasbeen shortened from 29 to 28 amino acids and the “shift to the left” ofamino acids kevl(i/v)wg (v/i)hh (SEQ ID NO: 367) is clearly evident.

In 2003, one Replikin was further shortened (or compacted) to the 21amino acid Replikin of the first listed 2003 human SARS virus. The % kof the 2003 SARS Replikin is now 38.1% (8/21) in comparison to 20.7% ofthe Goose Replikin and the 1918 Human Pandemic Replikin. Compared to theinfluenza 29 amino acid Replikin, three SARS Replikins were found to befurther shortened (or compacted) to 19, 11 and 9 amino acid longsequences, respectively. In the SARS 9 amino acid sequences shown, the %k is 44.4% (4/9). With the shortening of the SARS Replikin, the SARSmortality rate in humans rose to 10% in the young and 55.5% in theelderly compared to the 2.5% mortality in the 1918 influenza pandemic.

The amino acid sequences are shown in Table 7 to emphasize the degree ofhomology and conservation for 85 years (1917-2002) of the influenzaReplikin, for which evidence has first been observed in the 1917 GooseReplikin. No such conservation has ever been observed before. Table 7also illustrates that the Replikins in the 2003 human SARS virus, inaddition to having homologies to the influenza Replikins which firstappeared as the 1917 Goose Replikin and the 1918 Human Pandemicinfluenza Replikin, show certain sequence homologies to both thecoronavirus avian bronchitis virus Replikin (e.g. “k” in positions 1 and2, end in “h”) and to the coronavirus acute diarrhea virus Replikin(e.g. “k” in positions 1 and 11, “h” at the end of the Replikin). Thisevidence of relation to both influenza and coronavirus Replikins is ofinterest because SARS arose in Hong Kong as did several recent influenzaepidemics and earlier pandemics, and the SARS virus has been classifiedas a new coronavirus partly because of its structure, includingnucleocapsid, spike, and envelope proteins. Certain epidemiologicalevidence also is relevant in that SARS made its first appearance inhumans as the epidemic pneumonia, which erupted, in a crowded Hong Kongapartment house where there was a severe back-up of fecal sewage, whichwas airborne by ventilating fans.

Composition of Replikins in Strains of Influenza Virus B: Of a total of26 Replikins identified in this strain (Table 3), the following tenReplikins are present in every influenza B isolate examined from1940-2001. Overlapping Replikin sequences are listed separately. Lysinesand histidines are in bold type to demonstrate homology consistent withthe “3-point recognition.”

KSHFANLK (SEQ ID NO: 104) KSHFANLKGTK (SEQ ID NO: 105)KSHFANLKGTKTRGKLCPK (SEQ ID NO: 106) HEKYGGLNK (SEQ ID NO: 107)HEKYGGLNKSK (SEQ ID NO: 108) HEKYGGLNKSKPYYTGEHAK (SEQ ID NO: 10)HAKAIGNCPIWVK (SEQ ID NO: 110)HAKAIGNCPIWVVKKTPLKLANGTK (SEQ ID NO: 111) HAKAIGNCPIWVKTPLKLANGTKYRPPAK(SEQ ID NO: 112) HAKAIGNCPIWVKTPLKLANGTKYRPPAKLLK (SEQ ID NO: 113)

Tables 3 and 4 indicate that there appears to be much greater stabilityof the Replikin structures in influenza B hemagglutinins compared withH1N1 Replikins. Influenza B has not been responsible for any pandemic,and it appears not to have an animal or avian reservoirs. (Stuart-Harriset al., Edward Arnold Ltd., London (1985)).

Replikins in Influenza Over Time

Only one Replikin “hp(v/i)tigecpkyv-(r/k)(s/t)(t/a)k” (SEQ ID NO: 135)is present in every H1N1 isolate for which sequences are available from1918, when the strain first appeared and caused the pandemic of thatyear, through 2000. (Table 4). (“(v/i)” indicates that the amino acid vor i is present in the same position in different years.) Although H1N1contains only one persistent Replikin, H1N1 appears to be more prolificthan influenza B. There are 95 different Replikin structures in 82 yearson H1N1 versus only 31 different Replikins in 62 years of influenza Bisolates (Table 4). An increase in the number of new Replikin structuresoccurs in years of epidemics (Tables 3, 4, 5 and 6) and correlates withincreased total Replikin concentration (FIGS. 7, 8, 11 and 15).

Influenza H2N2 Replikins: Influenza H2N2 was responsible for the humanpandemic of 1957. Three of the 20 Replikins identified in that strainfor 1957 were conserved in each of the H2N2 isolates available forexamination on PubMed until 1995 (Table 5).

-   -   ha(k/q/m)(d/n)ilekthngk (SEQ ID NO: 232)    -   ha(k/q/m)(d/n)ilekthngklc(k/r) (SEQ ID NO: 233)    -   kgsnyp(v/i)ak(g/r)synntsgeqmliiwq(v/i)h (SEQ ID NO: 238)

However, in contrast to H1N1, only 13 additional Replikins have beenfound in H2N2 beginning in 1961. This paucity of appearance of newReplikins correlates with the decline in the concentration of the H2N2Replikins and the appearance of H2N2 in isolates over the years. (FIG.8).

Influenza H3N2 was responsible for the human pandemic of 1968. FiveReplikins which appeared in 1968 disappeared after 1977, but reappearedin the 1990s (Table 6). The only Replikin structure which persisted for22 years was hcd(g/q)f(q/r)nekwdlf(v/i)er(s/t)k (SEQ ID NO: 277), whichappeared first in 1977 and persisted through 1998. The emergence oftwelve new H3N2 Replikins in the mid 1990s (Table 6) correlates with theincrease in Replikin concentration at the same time (FIG. 8), and withthe prevalence of the H3N2 strain in recent isolates together with theconcurrent disappearance of all Replikins from some of these isolates(FIG. 8), this suggests the emergence of the new substrain H3N2(R). Thecurrent epidemic in November-December 2003 of a new strain of H3N2(Fujian) confirms this prediction made first in the ProvisionalApplication U.S. 60/303,396, filed Jul. 9, 2001.

FIGS. 7, 8, 11 and 15 show that influenza epidemics and pandemicscorrelate with the increased concentration of Replikins in influenzavirus, which is due to the reappearance of at least one Replikin fromone to 59 years after its disappearance. Also, in the A strain only,there is an emergence of new strain-specific Replikin compositions(Tables 4-6, see also increase in number of new Replikins, pre-epidemicfor H5N1 in FIGS. 11 and 15). Increase in Replikin concentration byrepetition of individual Replikins within a single protein appears notto occur in influenza virus, but is seen in other organisms.

It has been believed that changes in the activity of different influenzastrains are related to sequence changes in influenza hemagglutinins,which in turn are the products of substitutions effected by one of twopoorly understood processes: i) antigenic drift, thought to be due tothe accumulation of a series of point mutations in the hemagglutininmolecule, or ii) antigenic shift, in which the changes are so great thatgenetic reassortment is postulated to occur between the viruses of humanand non-human hosts. First, the present data suggests that the change inactivity of different influenza strains, rather than being related tonon-specific sequence changes, are based upon, or relate to theincreased concentration of strain-specific Replikins and strain-specificincreases in the replication associated with epidemics. In addition, thedata were examined for a possible insight into which sequence changesare due to “drift” or “shift”, and which are due to conservation,storage in reservoirs, and reappearance. The data show that theepidemic-related increase in Replikin concentration is not due to theduplication of existing Replikins per hemagglutinin, but is due to thereappearance of at least one Replikin composition from 1 to up to 59years after its disappearance, plus in the A strains only, the emergenceof new strain-specific Replikin compositions (Tables 3-6). Thus theincrease in Replikin concentration in the influenza B epidemics of 1951and 1977 are not associated with the emergence of new Replikincompositions in the year of the epidemic but only with the reappearanceof Replikin compositions which had appeared in previous years thendisappeared (Table 3). In contrast, for the A strains, in addition tothe reappearance of previously disappeared virus Replikins, newcompositions appear (e.g. in H1N1 in the year of the epidemic of 1996,in addition to the reappearance of 6 earlier Replikins, 10 newcompositions emerged). Since the A strains only, not influenza B, haveaccess to non-human animal and avian reservoirs, totally newcompositions probably derive from non-human host reservoirs rather thanfrom mutations of existing human Replikins which appear to bear noresemblance to the new compositions other than the basic requirements of“3-point recognition” (Tables 2-5). The more prolific nature of H1N1compared with B, and the fact that pandemics have been produced by thethree A strains only, but not by the B strain, both may also be afunction of the ability of the human A strains to receive new Replikincompositions from non-human viral reservoirs.

Some Replikins have appeared in only one year, disappeared, and notreappeared to date (Tables 3-6). Other Replikins disappear from one toup to 81 years, when the identical Replikin sequence reappears. KeyReplikin ‘k’ and ‘h’ amino acids, and the spaces between them, areconserved during the constant presence of particular Replikins over manyyears, as shown in Tables 2 and 3-6 for the following strain-specificReplikins: ten of influenza B, the single Replikin of H1N1, and thesingle Replikin of H3N2 as well as for the reappearance of identicalReplikins after an absence. Despite the marked replacement orsubstitution activity of other amino acids both inside the Replikinstructure and outside it in the rest of the hemagglutinin sequences,influenza Replikin histidine (h) appears never to be, and lysine (k) israrely replaced. Examples of this conservation are seen in the H1N1Replikin “hp(v/i)tigecpkyv(r/k)(s/t)(t/a)k,” (SEQ ID NO: 135) constantbetween 1918 and 2000, in the H3N2 Replikin“hcd(g/q)f(q,r)nekwdlf(v/i)er(s/t)k” (SEQ ID NO: 277) constant between1975 and 1998 and in the H3N2 Replikin“hqn(s/e)(e/q)g(t/s)g(q/y)aad(l/q)kstq(a/n)a(i/l)d(q/g)I(n/t)(g/n)k,(l/v)n(r/s)vi(e/c)k” (SEQ ID NO: 276) which first appeared in 1975, disappeared for25 years, and then reappeared in 2000. While many amino acids weresubstituted, the basic Replikin structure of 2 Lysines, 6 to 10 residuesapart, one histidine, a minimum of 6% lysine in not more thanapproximately 50 amino acids, was conserved.

Totally random substitution would not permit the persistence of theseH1N1 and H3N2 Replikins, nor from 1902 to 2001 in influenza B thepersistence of 10 Replikin structures, nor the reappearance in 1993 of a1919 18-mer Replikin after an absence of 74 years. Rather than a randomtype of substitution, the constancy suggests an orderly controlledprocess, or in the least, protection of the key Replikin residues sothat they are fixed or bound in some way: lysines, perhaps bound tonucleic acids, and histidines, perhaps bound to respiratory redoxenzymes. The mechanisms, which control this conservation, are at presentunknown.

H5N1 Influenza Conservation of Replikin Scaffold

There is concern that the current outbreak of high mortality H5N1 “birdflu” in several countries may represent the first phase of an overdueinfluenza pandemic. A recent report suggests that in the first probableperson-to-person transmission of H5N1, “sequencing of the viral genesidentified no change in the receptor-binding site of hemagglutinin orother key features of the virus. The sequences of all eight viral genesegments clustered closely with other H5N1 sequences from recent avianisolates in Thailand.” Phylogenetic analysis suggested that from theabsence of evidence of “reassortment with human influenza viruses”thatH5N1 is not a new variant. However, we now report three recent changesin a specific H5N1 protein sequence at sites which had not been changedin the last two H5N1 epidemics and in fact had been conserved since1959.

Previously, there has been no protein chemistry which correlated withvirus epidemics and dormancy. We found that each of the three influenzapandemics of the last century, H1N1, H2N2 and H3N2, retrospectively waspredicted by and correlated with an increase in the concentration of aspecific class of peptides in the virus, rich in lysine and histidine,associated with rapid replication, called replikins. We have now againfound the replikins to be predictive in each of the three H5N1epidemics, in 1997, 2001, and 2003-2004 (FIG. 15). Each year that theyappear in isolates, the replikins can now be counted per 100 amino acidsas in FIG. 15, and their sequences analyzed and compared as in Table 9.Analysis of replikins may be accomplished manually or in a preferredaspect of the present invention automatically by software designed bythe inventors for the purpose of counting replikin concentration inavailable sequence information.

A graph illustrating a rapid increase in the concentration of Replikinpatterns in the hemagglutinin protein of the H5N1 strain of influenzaprior to the outbreak of three “Bird Flu” epidemics may be seen in FIG.15. A review of FIG. 15 illustrates that an increasing replikinconcentration (‘Replikin Count’) in the hemagglutinin protein of H5N1preceded three ‘Bird Flu’ Epidemics. For example, an increase in theReplikin Count (Means±/−SD) in 1995 to 1997 preceded the Hong Kong H5N1epidemic of 1997 (E1). An increase in the Replikin Count from 1999 to2001 preceded the epidemic of 2001 (E2). And an increase in ReplikinCount from 2002 to 2004 preceded the epidemic in 2004 (E3). The declinein 1999 occurred with the massive culling of poultry in response to theE1 epidemic in Hong Kong.

In addition to the total number of replikins in the virus protein, thestructure of each replikin through time is informative. Table 8 shows areplikin first observed in a goose infected with influenza in 1917(Goose Replikin). Constant length, constant lysines at the aminoterminal and histidine residues at the carboxy terminal were conservedin different strains in a fixed scaffold for decades. Homologues of theGoose Replikin appeared from 1917 to 2006 in strains including eachresponsible for the three pandemics of 1918, 1957, and 19681, H1N1, H2N2and H3N2, and with further substitutions between H1N2, H7N7, H5N2 andH5N1. Even certain substitutions which have occurred in the GooseReplikin tend to be selective and retained for years, rather thanrandom. Thus despite the common assumption that amino acid substitutionsshould occur at random, it would appear that not all substitutions ininfluenza are, in fact, random. This replikin conservation over decadesallows the production of synthetic influenza vaccines which rapidly andinexpensively can be prepared in advance and can be effective for morethan one year.

Therefore a target for synthetic influenza vaccines is the conservedReplikin Scaffold in influenza virus. A Replikin Scaffold comprises aseries of conserved peptides comprising a sequence of about 16 to about30 amino acids and further comprising

-   -   (1) a terminal lysine;    -   (2) a terminal histidine and another histidine in the residue        portion immediately adjacent to the terminal histidine;    -   (3) at least one lysine within about 6 to about 10 amino acid        residues from at least one other lysine; and    -   (4) at least about 6% lysines within the 16 to about 30 amino        acid peptide.        A Replikin Scaffold may further comprise a an additional lysine        immediately adjacent to the terminal lysine. “Replikin Scaffold”        peptides may comprise an additional lysine immediately adjacent        to the terminal lysine. “Replikin Scaffold” peptide also refers        to an individual member or a plurality of members of a series of        a “Replikin Scaffold.”

A non-limiting and preferred target for synthetic influenza vaccines maybe a Replikin Scaffold in influenza virus further comprising a sequenceof about 29 amino acids and a lysine immediately adjacent to theterminal lysine.

A non-preferred target for synthetic influenza may be an ExoskeletonScaffold in a first strain of influenza virus comprising a first peptideof about 29 amino acids and

-   -   (1) a terminal lysine and a lysine immediately adjacent to the        terminal lysine;    -   (2) a terminal histidine and a histidine immediately adjacent to        the terminal histidine;    -   (3) no lysine within 6 to 10 amino acid residues from any other        lysine        wherein an earlier-arising specimen of the first strain or        another strain of virus comprises a Replikin Scaffold of about        29 amino acids.

In the 1997 H5N1 Hong Kong epidemic, the human mortality rate wasapproximately 27%. In 2004, of the fifty-two people reported to havebeen infected by H5N1 in Asia approximately 70% died. Most recently,nine of the eleven cases in Vietnam from Dec. 28, 2004 to Jan. 27, 2005died. Although the virulence of the virus appears to have increased, anychanges thought to be required for further spread human to human, hadbeen thought not yet to have occurred 1. However, we now have observedrecent substitutions in three H5N1 replikin amino acid residues atposition numbers 18, 24 and 28 of the Goose Replikin scaffold fromisolates in Vietnam, Thailand and China in 2004 (see Table 1).Substitution at site number 24 has not occurred since the appearance ofH5N1 in 1959 but was present in the last two influenza pandemics causedby other strains, H2N2 in 1957 and H3N2 in 1968, together responsiblefor over two million human deaths, and in a recent virulent epidemiccaused by H7N7 (see Table 8). While these are only hints of possibledanger, these data on substitution, combined with the rising ReplikinCount shown in FIG. 15, and the past correlation of such replikin datawith pandemics, does not give the same reassurance as that obtained fromphylogenetic analysis that the virus is unlikely to spread human tohuman.

With respect to the H5N1 influenza, FIG. 15 illustrates a rapid increasein the concentration of Replikins per 100 amino acids just prior toepidemics in 1997 (indicated as E1), 2001 (indicated as E2) and 2004(indicated as E3).

TABLE 8Replikin Scaffold showing ordered substitution in the 89 year conservationof influenza virus replikin peptides related to rapid replication, from a 1917goose influenza replikin and the 1918 human pandemic replikin to 2006H5N1 “Bird Flu” homologues.(SEQ ID NOS: 368-429, respectively, in order of appearance)[<------29 Amino Acids------>] Year Strain kkgtsypklsksytnnkgkevlvlwgvhh1917 H1N_ Influenza Goose Replikin kkgssypklsksyvnnkgkevlvlwgvhh 1918H1N1 Human Influenza Pandemic kk ensypklsksyvnnkgkevlvlwgvhh 1930 H1N1kkgdsypkltnsyvnnkgkevlvlwgvhh 1933 H0N1 kkgtsypklsksytnnkgkevlvlwgvhh1976 H1N1 kkgtsypklsksytnnkgkevlvlwgvhh 1977 H1N1kkgnsypklsksytnnkgkevlv i wgvhh 1979 H1N1 kkgnsypklsksytnnkgkevlv iwgvhh 1980 H1N1 kkgtsypklsksytnnkgkevlvlwgvhh 1980 H1N1kkgnsypklsksytnnkgkevlv i wgvhh 1981 H1N1 kkgtsypklsksytnnkgkevlvlwgvhh1981 H1N1 kkgtsypklsksytnnkgkevlvlwgvhh 1985 H1N1kkgnsypklsksytnnkgkevlv i wgvhh 1991 H1N1 kkgnsypklsksytnnkgkevlv iwgvhh 1992 H1N1 kkgnsypklsksytnnkgkevlv i wgvhh 1996 H1N1kkgdsypklsksytnnkgkevlv i wgvhh 1996 H1N1 kkgssypklsksyvnnkgkevlvlwgvhh1997 H1N1 kkgssypklsksyvnnkgkevlvlwgvhh 1998 H1N1kkgnsypklsksytnnkgkevlv i wgvhh 1999 H1N1 kkgnsypklsksytnnkgkevlv iwgvhh 2000 H1N1 kkgnsypklsksytnnkgkevlv i wgvhh 2001 H1N1kkgnsypklsksytnnkgkevlv i wgvhh 2002 H1N1 kkgnsypk isksyinnk e kevlvlwgihh 1999 H1N2 Influenza kkgnsypklsksyinnkkkevlv i wgi hh 2000 H1N2kkgnsypklsksyinnkgk k vlvlwgi hh 2001 H1N2 kkgtsypklsksytnnk kkevlvlwgvhh 2001 H1N2 -k nglypnlsksyannk e kevlvlwgvhh 2002 H1N2 -knglypnlsksyannk e kevlilwgvhh 2002 H1N2 kkgpnypva k rsynntsgeqmli iwgvhh 1957 H2N2 Human Influenza Pandemic kkgpnypva k rsynntsgeqmli i wgihh 1957 H2N2 Human Influenza Pandemic kk ensypklr ksiiink k ev k lv iwgi hh 1968 H3N2 Human Influenza Pandemic ----------ksy k ntr k dpali iwgi hh 1979-2003 H7N7 Influenza kk nnaypti k rtynntnvedllilwgi hh 2002H5N2 Influenza kk nnaypti k rsysntnqedllvlwgi hh 1959H5N1 Influenza (Scotland) kk nnaypti k rtynntniedllilwgi hh 1975H5N1 (Wisconsin) kk nnaypti k rtynntnmedllilwgi hh 1981 H5N1 (Minnesota)kkgnaypti k rtynntnvedllilwgi hh 1983 H5N1 (Pennsylvania) kk nntypti krsynntnqedllilwgi hh 1988 H5N1 (Scotland) kk nsaypti k rsynntnqedllvlwgihh 1996 H5N1 (China) kk nsaypti k rsynntnqedllvlwgi hh 1997 H5N1 (China)kk nsaypti k rsynntnqedllvlwgi hh 1998 H5N1 (China) kk nsaypti krsynntnqedllvlwgi hh 1999 H5N1 (China) kknsaypti k rsynntnqedllvlwgi hh2000 H5N1 (China) kk nsaypti k rsynntnqedllvlwgi hh 2001 H5N1 (China) kknnayptikrsynntnqedllvlwgi hh 2001 H5N1 (China) kknsayptikrsynntnqedllvlwgi hh 2002 H5N1 (China) kknstyptikrsynntnqedllvlwgi hh 2002 H5N1 (Thailand) kknstyptikrsynntnqedllvlwgi hh 2002 H5N1 (Vietnam) kknstyptikrsynntnqedllvlwgi hh 2003 H5N1 (Vietnam) kknstyptikrsynntnqedllvlwgi hh 2003 H5N1 (Thailand) kknstyptikrsynntnqedllvlwgi hh 2003 H5N1 (Sindong, China) kknnayptikrsynntnqedllvlwgi hh 2003 H5N1 (China) kk nstyptikrsynntnqedllvm wgi hh 2004 H5N1 (Vietnam, highly pathogenic) kknsayptikrsynntnqedllvlwgi hh 2004H5N1 (Vietnam, “highly pathogenic”, gull) kk nstyptikrsynntnqedllvlwgihh 2004 H5N1 (Vietnam, highly pathogenic) kk nstyptikrsynntnqedllvlwgihh 2004 H5N1 (Thailand, highly pathogenic) kk nstypti krsynntnqedllvlwgi q h 2004 H5N1 (Thailand, highly pathogenic) kk nsaypiik rsynntnqedllvlwgi hh 2004 H5N1 (China, highly pathogenic) kk nsaypti krsxnntn h edllvlwgi hh 2004 H5N1 (China, “highly pathogenic”, goose) kknsaypti k rsynntnqedllvlwgi hh 2004 H5N1 Japan kk nnaypti krsynntnqedllvlwgi hh 2005 H5N1 Turkey kk nntypti k ksynntnqedllvlwgi hh2006 H5N1 China (Anhui) * Residues identical to Goose Replikin aminoacids un-underlined; amino acid substitutions underlined and italicizedto show scaffold pattern across years and strains.

Table 8, above, provides further support for the role of replikins inepidemics and pandemics in humans and birds. In Table 8, the history ofthe Goose Replikin and its homologues are tracked from 1917 to thepresent outbreak of avian H5N1 virus. Table 8 demonstrates conservationof the “scaffold” homology of the Goose Replikin in virulent strains ofinfluenza.

Table 8 illustrates the history, by year or smaller time period, of theexistence in the protein structure of the Goose Replikin and itshomologues in other influenza Replikins. Table 8 further illustrates thehistory of amino acid substitutions in those homologues and theconservation of certain amino acids of the Replikin structure which areessential to the definition of a Replikin and the function of rapidreplication supplied by Replikins.

A review of Table 8 illustrates that if random substitution of aminoacids were to occur in virulent strains of influenza from 1917 throughthe present, certain framework amino acids of the Goose Replikin wouldnot be conserved from year to year in strains in which epidemicsoccurred. However, contrary to what would result from randomsubstitution, virulent strains of influenza from year to yearconsistently contain conserved amino acids at those positions thatdefine a Replikin. That is, if a substitution were to occur in one ofthe amino acids that define a Replikin, e.g. lysine or a histidine, thedefinition of the Replikin would be lost. Nevertheless, the Replikinsequence is conserved over more than 85 years. Thus, since there isconservation of certain amino acids over decades, substitution cannot besaid to be completely at random. The fact that substitutions do occur inamino acids that are not essential to the definition of a Replikin(i.e., amino acids other than lysines or histidines) demonstrates theimportance of the Replikin in the pathogenicity of the strain.

It may be further noted from Table 8 that when substitutions do occur,they are seen to occur at certain apparently preferred positions of theReplikin Scaffold. Table 8 illustrates recurring substitutions atpositions 1, 3-24 and 26-27. Further, while substitutions occurthroughout these positions, a lysine continues to exist at a position 6to 10 amino acids from the second lysine (which has not been substitutedin these virulent strains).

Even when there is a substitution of a lysine position within the 29amino acid stretch, as is seen in 1957, when K at position 11 shifts toposition 10, that new position has been maintained until 2005, as haveYP, AY, N (position 15), and LVLWG (SEQ ID NO: 430) to conserve thehomologous structure of the Replikin Scaffold with few exceptions.

Table 8 demonstrates the integrity of the Replikin Scaffold in virulentstrains of influenza. As discussed above, degeneration of the ReplikinScaffold into an Exoskeleton Scaffold is seen to decrease pathogenicity.The integrity and conservation of the Replikin Scaffold, therefore, isseen by the fact that there is generally a fixed 29 amino acid sequencethat begins with two lysines and ends with two histidines.

It is important to note that an extra K has appeared in the ReplikinScaffold of a 2006 strain of H5N1 in China (Anhui). This presence of anextra K signals an increase in the Replikin count within the ReplikinScaffold. The 2006 China (Anhui) strain has a Replikin count of 6.6 (asdiscussed below). A Replikin count of 6.6 is the highest ever observedfor an H5N1 strain and is comparable in the entire A strain of influenzaonly to the Replikin count of the influenza strain that caused the 1918Pandemic. If this initial 2006 report is repeated and maintained, it mayindicate that the Counts of 4.5 and 4.0 in 2004 and 2005 respectivelywill be substantially increased, and foretell a continuing or increasedepidemic of H5N1 ‘Bird Flu’.

An aspect of the present invention is a combination of replikinstructure and function to track the pathogenicity or rate of replicationof a virus, epidemic or pandemic or to predict the occurrence ofepidemics or pandemics. An example of this combination is the ability ofthe Replikin algorithm of the invention to be used to count increases inReplikin counts in influenza strains such as the strain of 1918 and thecurrent H5N1 strain of H5N1. The Replikin Count of the 1918 influenzapandemic and the current outbreak of “Bird Flu” demonstrate thepredictive capacity of this exemplary aspect in accordance with and madepossible by the invention.

Relation of Some Shrimp White Spot Virus Replikins to Influenza FixedScaffold Replikin Structures

The inventors have also established a relationship between virulentinfluenza virus and white spot virus in the Replikin Scaffold portionsof the viruses. No relationship between these two viruses has beensuggested previously. Although there is extensive substitution, theapplicants' finding of several short Replikins of the Shrimp White SpotSyndrome Virus demonstrate significant homologies to the influenza virusReplikin sequences, especially with regard to length and key lysine (k)and histidine (h) residues (Fixed Scaffold or Replikin Scaffold),suggesting that similar mechanisms of Replikin production are used inboth virus groups.

TABLE 8A Shrimp White Spot Scaffolding(SEQ ID NOS: 431-440, respectively, in order of appearance)Kkgtsypklsksytnnkgkevlvlwgvhh 1917 HIN_ Influenza goose peptideKkgnsypklsksytnnkgkevlv i wgvhh 2002 H1N1 Swine Influenza Kknv k sa kqlp h l k yl k k ldvrga k qlp h 2000 Shrimp White Spot Syndrome Virus -kv h ldv k gv k qll h l k vrldvrga k ql h 2000 Shrimp White Spot SyndromeVirus kk ensypklr ksiiink k ev k lv i wgi hh 1968 H3N2 Human Influenza----------ksy k ntr k dpali i wgihh 1979-2003 Pandemic H7N7 Influenzakkgpnypva k rsynntsgeqmli i wgvhh 1957 H2N2 Human Influenza Pandemickkgpnypva k rsynntsgeqmli i wgi hh 1957 H2N2 Human Influenza Pandemic kknnayptikrtynntnvedllilwgi hh 2002 H5N2 Influenza kknnayptikrsysntnqedllvlwgi hh 1959 H5N1 Influenza Residues identical tooriginal 1917 Goose Replikin residues are shown un-underlined. Aminoacid substitutions are shown underlined and in italics.

In addition, since many species, including but not limited to swine andbirds, are known to provide animal “reservoirs” for human influenzainfection, marine forms such as the shrimp virus can now be examined,with early warning diagnostic benefits possible for outbreaks such asswine flu and bird flu. While similarities of some influenza viruseswere noted between species, and the transfer of these virusesinterspecies was known, there was no previous quantitative method togauge virus activity. It has not been possible previously to examinepotential reservoirs for increased activity which might move into adifferent species; thus providing an advanced warning. The activity ofthe Replikins in each species can now be monitored constantly forevidence of increased viral replication rate and thus emergence ofepidemics in that species which may then transfer to other species.

This data further supports the Replikins as a new class of peptides,with a history of its own, and a shared function of rapid replicationand disease of its hosts. With the high mortality for its shrimp host,white spot syndrome virus can now have its Replikins examined as earlierforms of the virus Replikins, or as parallel morphological branches,which in either case may well act as reservoirs for bird and animalReplikins such as those in influenza viruses. The diagnostic andpreventive uses of these Replkin findings in shrimp follow as they do ininfluenza and for other organisms containing Replikins.

Conservation of Replikin Structures

Whether Replikin structures are conserved or are subject to extensivenatural mutation also was examined by scanning the protein sequences ofvarious isolates of foot and mouth disease virus (FMDV), where mutationsin proteins of these viruses have been well documented worldwide fordecades. Protein sequences of FMDV isolates were visually examined forthe presence of both the entire Replikin and each of the componentReplikin amino acid residues observed in a particular Replikin.

Rather than being subject to extensive substitution over time as occursin neighboring amino acids, the amino acids which comprise the Replikinstructure are substituted little or not at all, that is the Replikinstructure is conserved.

For example, in the protein VP1 of FMDV type O, the Replikin (SEQ ID NO:3) “hkqkivapvk” was found to be conserved in 78% of the 236 isolatesreported in PubMed, and each amino acid was found to be conserved inindividual isolates as follows: his, 95.6%; lys, 91.8%; gln 92.3%; lys,84.1%; ile, 90.7%; val, 91.8%; ala, 97.3%; pro, 96.2%; ala, 75.4%; andlys, 88.4%. The high rate of conservation suggests structural andfunctional stability of the Replikin structure and provides constanttargets for treatment.

Similarly, sequence conservation was found in different isolates of HIVfor its Replikins, such as (SEQ ID NO: 5) “kcfncgkegh” or (SEQ ID NO: 6)“kvylawvpahk” in HIV Type 1 and (SEQ ID NO: 7) “kcwncgkegh” in HIV Type2 (Table 2). Further examples of sequence conservation were found in theHIV tat proteins, such as (SEQ ID NO: 441) “hclvckqkkglgisygrkk,”wherein the key lysine and histidine amino acids are conserved. (SeeTable 9).

Similarly, sequence conservation was observed in plants, for example inwheat, such as in wheat ubiquitin activating enzyme E (SEQ ID NOs.454-456). The Replikins in wheat even provided a reliable target forstimulation of plant growth as described within. Other examples ofconservation are seen in the constant presence of malignin in successivegenerations, over ten years of tissue culture of glioma cells, and bythe constancy of affinity of the glioma Replikin for antimaligninantibody isolated by immunoadsorption from 8,090 human sera from theU.S., U.K., Europe and Asia (e.g., FIG. 5 and U.S. Pat. No. 6,242,578B1).

Similarly, conservation was observed in trans-activator (Tat) proteinsin isolates of HIV. Tat (trans-activator) proteins are early RNA bindingproteins regulating lentiviral transcription. These proteins arenecessary components in the life cycle of all known lentiviruses, suchas the human immunodeficiency viruses (HIV). Tat is a transcriptionalregulator protein that acts by binding to the trans-activating responsesequence (TAR) RNA element and activates transcription Initiation and/orelongation from the LTR promoter. HIV cannot replicate without tat, butthe chemical basis of this has been unknown. In the HIV tat proteinsequence from 89 to 102 residues, we have found a Replikin that isassociated with rapid replication in other organisms. The amino acidsequence of this Replikin is “HCLVCKQKKGLGISYGRKK.” (SEQ ID NO: 441) Infact, we found that this Replikin is present in every HIV tat protein.Some tat amino acids are substituted frequently by alternate amino acids(in small size fonts lined up below the most frequent amino acid (Table9), the percentage of conservation for the predominant Replikin“HCLVCFQKKGLGISYGRKK” (SEQ ID NO: 442)). These substitutions haveappeared for most of the individual amino acids. However, the key lysineand histidine amino acids within the Replikin sequence, which define theReplikin structure, are conserved 100% in the sequence; whilesubstitutions are common elsewhere in other amino acids, both within andoutside the Replikin, none occurs on these key histidine amino acids.

As shown in Table 9 it is not the case that lysines are not substitutedin the tat protein amino acid sequence. From the left side of the table,the very first lysine in the immediate neighboring sequence, but outsidethe Replikin sequence, and the second lysine (k) in the sequence insidethe Replikin, but “extra” in that it is not essential for the Replikinformation, are both substituted frequently. However, the 3rd, 4th and5th lysines, and the one histidine, in parentheses, which together setup the Replikin structure, are never substituted. Thus, these key aminoacid sequences are 100% conserved. As observed in the case of theinfluenza virus Replikins, random substitution would not permit thisselective substitution and selective non-substitution to occur due tochance.

TABLE 9% Replikin CONSERVATION of each constituent amino acid in the first 117different isolates of HIV tat protein as reported in PubMed: 38 (100) 57 86 (100) (100) 66 76 (100) 99 57 49 (100) 94 (100) 97 98 85 97 99 (100)(100)(100)%Neighboring Amino acids                       tat Replikink (c) s y [(h) (c)  l v (c) f q k (k) g  (l) g i s y g  (r)(k)(k)]below are the amino acid substitutions observed for each amino acid above: h     c f        q  i       l h t       a        a l y h qr     w p        l  l         i h       q        vy       s           s         l m       r        si                   s         m s s                   r         n v a fp q

The conservation of the Replikin structure suggests that the Replikinstructure has a specific survival function for the HIV virus which mustbe preserved and conserved, and cannot be sacrificed to the virus‘defense’ maneuver of amino acid substitution created to avoid antibodyand other ‘attack.’ These ‘defense’ functions, although also essential,cannot ‘compete’ with the virus survival function of HIV replication.

Further conservation was observed in different isolates of HIV for itsReplikins such as “kcfncgkegh” (SEQ ID NO: 5) or “kvylawvpahk” (SEQ IDNO: 6) in HIV Type 1 and “kcwncgkegh” (SEQ ID NO: 7) in HIV Type 2. Thehigh rate of conservation observed in FMVD and HIV Replikins suggeststhat conservation also observed in the Replikins of influenza Replikinsis a general property of viral Replikins. This conservation makes them aconstant and reliable targeted for either destruction, for example byusing specific Replikins such as for influenza, FMVD or HIV vaccines asillustrated for the glioma Replikin, or stimulation.

Similarly, as provided in examples found in viruses including influenzaviruses, FMDV, and HIV, where high rates of conservation in Replikinssuggest that conservation is a general property of viral Replikins andthus making Replikins a constant and reliable target for destruction orstimulation, conservation of Replikin structures occurs in plants. Forexample, in wheat plants, Replikins are conserved and provide a reliabletarget for stimulation. Examples of conserved Replikins in wheat plantsubiquitin activating enzyme E include:

E3 HKDRLTKKVVDIAREVAKVDVPEYRRH (SEQ ID NO: 454)E2 HKERLDRKVVDVAREVAKVEVPSYRRH (SEQ ID NO: 455)E1 HKERLDRKVVDVAREVAKMEVPSYRRH (SEQ ID NO: 456)     * *     *  ** *

Similarly to conservation found in the HIV tat protein, the Replikin inthe wheat ubiquitin activating enzyme E is conserved. As with the HIVtat protein, substitutions of amino acids (designated with an ‘*’)adjacent to the Replikin variant forms in wheat ubiquitin activatingenzyme E are common. The key k and h amino acids that form the Replikinstructure, however, do not vary whereas the ‘unessential’ k that is only5 amino acids (from the first k on the left) is substituted.

Anti-Replikin Antibodies

An anti-Replikin antibody is an antibody against a Replikin. Data onanti-Replikin antibodies also support Replikin class unity. Ananti-Replikin antibody response has been quantified by immunoadsorptionof serum antimalignin antibody to immobilized malignin (see Methods inU.S. Pat. No. 5,866,690). The abundant production of antimaligninantibody by administration to rabbits of the synthetic version of the16-mer peptide whose sequence was derived from malignin, absentcarbohydrate or other groups, has established rigorously that thispeptide alone is an epitope, that is, provides a sufficient basis forthis immune response (FIG. 3). The 16-mer peptide produced both IgM andIgG forms of the antibody. Antimalignin antibody was found to beincreased in concentration in serum in 37% of 79 cases in the U.S. andAsia of hepatitis B and C, early, in the first five years of infection,long before the usual observance of liver cancer, which develops aboutfifteen to twenty-five years after infection. Relevant to bothinfectious hepatitis and HIV infections, transformed cells may be oneform of safe haven for the virus: prolonging cell life and avoidingvirus eviction, so that the virus remains inaccessible to anti-viraltreatment.

Because administration of Replikins stimulates the immune system toproduce antibodies having a cytotoxic effect, peptide vaccines based onthe particular influenza virus Replikin or group of Replikins observedto be most concentrated over a given time period provide protectionagainst the particular strain of influenza most likely to cause anoutbreak in a given influenza season, e.g., an emerging strain orre-emerging strain For example, analysis of the influenza virushemagglutinin amino acid sequence on a yearly or bi-yearly basis,provides data which are useful in formulating a specifically targetedinfluenza vaccine for that year. It is understood that such analysis maybe conducted on a region-by-region basis or at any desired time period,so that strains emerging in different areas throughout the world can bedetected and specifically targeted vaccines for each region can beformulated.

Influenza Vaccines, Treatments and Therapeutics

Currently, vaccine formulations for influenza are changed twice yearlyat international WHO and CDC meetings. Vaccine formulations are based onserological evidence of the most current preponderance of influenzavirus strain in a given region of the world. However, prior to thepresent invention there has been no correlation of influenza virusstrain specific amino acid sequence changes with occurrence of influenzaepidemics or pandemics.

The observations of specific Replikins and their concentration ininfluenza virus proteins provides the first specific quantitative earlychemical correlates of influenza pandemics and epidemics and providesfor production and timely administration of influenza vaccines tailoredspecifically to treat the prevalent emerging or re-emerging strain ofinfluenza virus in a particular region of the world. By analyzing theprotein sequences of isolates of strains of influenza virus, such as thehemagglutinin protein sequence, for the presence, concentration and/orconservation of Replikins, influenza virus pandemics and epidemics canbe predicted. Furthermore, the severity of such outbreaks of influenzacan be significantly lessened by administering an influenza peptidevaccine based on the Replikin sequences found to be most abundant orshown to be on the rise in virus isolates over a given time period, suchas about one to about three years.

An influenza peptide vaccine of the invention may include a singleReplikin peptide sequence or may include a plurality of Replikinsequences observed in influenza virus strains. Preferably, the peptidevaccine is based on Replikin sequence(s) shown to be increasing inconcentration over a given time period and conserved for at least thatperiod of time. However, a vaccine may include a conserved Replikinpeptide(s) in combination with a new Replikin(s) peptide or may be basedon new Replikin peptide sequences. The Replikin peptides can besynthesized by any method, including chemical synthesis or recombinantgene technology, and may include non-Replikin sequences, althoughvaccines based on peptides containing only Replikin sequences arepreferred. Preferably, vaccine compositions of the invention alsocontain a pharmaceutically acceptable carrier and/or adjuvant.

The influenza vaccines of the present invention can be administeredalone or in combination with antiviral drugs, such as gancyclovir;interferon; interleukin; M2 inhibitors, such as, amantadine,rimantadine; neuraminidase inhibitors, such as zanamivir andoseltamivir; and the like, as well as with combinations of antiviraldrugs.

The influenza vaccine of the present invention may be administered toany animal capable of producing antibodies in an immune response. Forexample, the influenza vaccine of the present invention may beadministered to a rabbit, a chicken, a pig or a human. Because of theuniversal nature of replikin sequences, an influenza vaccine of theinvention may be directed at a range of strains of influenza or aspecific strain of influenza.

In a non-limiting aspect in accordance with the present invention, aninfluenza vaccine may be directed to an immune response against animalor human strain of influenza including influenza B, (A)H1N1, (A)H2N2 and(A)H3N2, or any human variant of the virus that may arise hereafter, aswell as strains of influenza predominantly in animals such as thecurrent avian H5N1. An influenza vaccine may further be directed to aparticular replikin amino acid sequence in any portion of an influenzaprotein.

In a non-limiting aspect in accordance with the present invention, aninfluenza vaccine may comprise a Replikin Scaffold of the H5N1 virussuch as KKNSTYPTIKRSYNNTNQEDLLVLWGIHH (SEQ ID NO: 15). In a furthernon-limiting aspect, an influenza vaccine may comprise a UTOPE such asKKKKH (SEQ ID NO: 457) or KKKKHKKKKKH (SEQ ID NO: 458). In a furtheralternative, a vaccine may comprise the addition of an adjuvant such asthe well known key limpet hemocyanin denoted with the abbreviation -KLH.In yet a further preferred non-limiting aspect, an influenza vaccine maycomprise a Replikin Scaffold of influenza H5N1 further comprising twoUTOPES and an adjuvant sequence such asKKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK (SEQ ID NO: 16)-KLH (denotinga key limpet hemocyanin adjuvant) (Vaccine V120304U2). An aspect of thepresent invention may comprise the Replikin Scaffold previouslyconstructed and shown in Table 8 as one of the Bird Flu Replikinslabelled“2004H5N1 Vietnam, highly pathogenic.” With administration of100 ug of the peptide of Vaccine V120304U2 injected subcutaneously intorabbits and chickens an antibody response was observed from unvaccinateddilutions of less than 1:50 to reach a peak in the third to fourth weekafter vaccination of from a dilution of 1:120,000 to greater than1:240,000. (See Example 7.)

Repetition and Overlapping Replikin Structures

Analysis of the primary structure of a Plasmodium farciparum malariaantigen located at the merozoite surface and/or within theparasitophorous vacuole revealed that this organism, like influenzavirus, also contains numerous Replikins. However, there are severaldifferences between the observation of Replikins in Plasmodiumfalciparum and influenza virus isolates. For example, Plasmodiumfalciparum contains several partial Replikins. Another difference seenin Plasmodium falciparum is a frequent repetition of individual Replikinstructures within a single protein, which was not observed withinfluenza virus. Repetition may occur by (a) sharing of lysine residuesbetween Replikins, and (b) by repetition of a portion of a Replikinsequence within another Replikin sequence.

High Concentrations of Replikin Correlates with Rapid Replication

Tomato leaf curl Gemini virus has devastated tomato crops in China andin many other parts of the world. Its replikins reach high countsbecause of overlapping replikins as illustrated below in a virusisolated in Japan where the replikin count was 20.7

The relationship of higher Replikin concentration to rapid replicationis also confirmed by analysis of HIV isolates. It was found that theslow-growing low titer strain of HIV (NSI, “Bru,” which is prevalent inearly stage HIV infection) has a Replikin concentration of 1.1 (±1.6)Replikins per 100 amino acids, whereas the rapidly-growing high titerstrain of HIV (SI, “Lai”, which is prevalent in late stage HIVinfection) has a Replikin concentration of 6.8 (±2.7) Replikins per 100amino acid residues.

Passive Immunity

In another aspect of the invention, isolated Replikin peptides may beused to generate antibodies, which may be used, for example to providepassive immunity in an individual. Passive immunity to the strain ofinfluenza identified by the method of the invention to be the mostlikely cause of future influenza infections may be obtained byadministering antibodies to Replikin sequences of the identified strainof influenza virus to patients in need. Similarly, passive immunity tomalaria may be obtained by administering antibodies to Plasmodiumfalciparum Replikin(s).

Various procedures known in the art may be used for the production ofantibodies to Replikin sequences. Such antibodies include but are notlimited to polyclonal, monoclonal, chimeric, humanized, single chain,Fab fragments and fragments produced by an Fab expression library.Antibodies that are linked to a cytotoxic agent may also be generated.Antibodies may also be administered in combination with an antiviralagent. Furthermore, combinations of antibodies to different Replikinsmay be administered as an antibody cocktail.

For the production of antibodies, various host animals or plants may beimmunized by injection with a Replikin peptide or a combination ofReplikin peptides, including but not limited to rabbits, mice, rats, andlarger mammals.

Monoclonal antibodies to Replikins may be prepared by using anytechnique that provides for the production of antibody molecules. Theseinclude but are not limited to the hybridoma technique originallydescribed by Kohler and Milstein, (Nature, 1975, 256:495-497), the humanB-cell hybridoma technique (Kosbor et al., 1983, Immunology Today,4:72), and the EBV hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Inaddition, techniques developed for the production of chimeric antibodies(Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) or othertechniques may be used. Alternatively, techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce Replikin-specific single chain antibodies.

Particularly useful antibodies of the invention are those thatspecifically bind to Replikin sequences contained in peptides and/orpolypeptides of influenza virus. For example, antibodies to any ofpeptides observed to be present in an emerging or re-emerging strain ofinfluenza virus and combinations of such antibodies are useful in thetreatment and/or prevention of influenza. Similarly, antibodies to anyReplikins present on malaria antigens and combinations of suchantibodies are useful in the prevention and treatment of malaria.

Antibody fragments which contain binding sites for a Replikin may begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecules and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries can be generated (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

The fact that antimalignin antibody is increased in concentration inhuman malignancy (see FIG. 5), regardless of cancer cell type, and thatthis antibody binds to malignant cells regardless of cell type now maybe explained by the presence of the Replikin structures herein found tobe present in most malignancies (FIG. 1 and Table 2). Population studieshave shown that antimalignin antibody increases in concentration inhealthy adults with age, and more so in high-risk families, as thefrequency of cancer increases. An additional two-fold or greaterantibody increase, which occurs in early malignancy, has beenindependently confirmed with a sensitivity of 97% in breast cancers 1-10mm in size. Shown to localize preferentially in malignant cells in vivo,histochemically the antibody does not bind to normal cells butselectively binds to (FIGS. 4A,B) and is highly cytotoxic to transformedcells in vitro (FIGS. 4C-F). Since in these examples the same antibodyis bound by several cell types, that is, brain glioma, hematopoieticcells (leukemia), and small cell carcinoma of lung, malignant Replikinclass unity is again demonstrated.

Antimalignin does not increase with benign proliferation, butspecifically increases only with malignant transformation andreplication in breast in vivo and returns from elevated to normal valuesupon elimination of malignant cells (FIG. 5). Antimalignin antibodyconcentration has been shown to relate quantitatively to the survival ofcancer patients, that is, the more antibody, the longer the survival.Taken together, these results suggest that anti-Replikin antibodies maybe a part of a mechanism of control of cell transformation andreplication. Augmentation of this immune response may be useful in thecontrol of replication, either actively with synthetic Replikins asvaccines, or passively by the administration of anti-Replikinantibodies, or by the introduction of non-immune based organic agents,such as for example, carbohydrates, lipids and the like, which aresimilarly designed to target the Replikin specifically.

In another aspect of the invention, immune serum containing antibodiesto one or more Replikins obtained from an individual exposed to one ormore Replikins may be used to induce passive immunity in anotherindividual or animal. Immune serum may be administered via i.v. to asubject in need of treatment. Passive immunity also can be achieved byinjecting a recipient with preformed antibodies to one or moreReplikins. Passive immunization may be used to provide immediateprotection to individuals who have been exposed to an infectiousorganism. Administration of immune serum or preformed antibodies isroutine and the skilled practitioner can readily ascertain the amount ofserum or antibodies needed to achieve the desired effect.

Synthetic Replikin Vaccines (Active Immunity)

Synthetic Replikin vaccines, based on Replikins such as the gliomaReplikin (SEQ ID NO: 1) “kagvaflhkk” or the hepatitis C Replikin (SEQ IDNO: 18) “hyppkpgcivpak”, or HIV Replikins such as (SEQ ID NO: 5)“kcfncgkegh” or (SEQ ID NO: 6) “kvylawvpahk” or preferably, an influenzavaccine based on conserved and/or emerging or re-emerging Replikin(s)over a given time period may be used to augment antibody concentrationin order to lyse the respective virus infected cells and release virusextracellularly where chemical treatment can then be effective.Similarly, a malaria vaccine, based on Replikins observed in Plasmodiumfalciparum malaria antigens on the merozoite surface or within theparasitophorous vacuole, for example, can be used to generate cytotoxicantibodies to malaria. Table 7 shows the relation of shortening orcompacting of Replikin sequences to mortality rate caused by theorganisms which contain these Replikins, to as short as seven aminoacids. This correlation has been found by us to be a general phenomenonregardless of the type of organism. We have also found that there may bea progression over time to the shortened Replikin structure, as ininfluenza and SARS viruses.

There is abundant evidence that there are constant evolutionary andcompetitive pressures for the emergence of constantly increasing“efficacy” of each infectious organism. Based upon these observations,and by projection, it would appear that if evolutionary pressures aretowards shorter and shorter Replikins, with higher and higherconcentrations of lysine (k), to as high as 70% as in EEL leukemia(Table 7), then the projected theoretical ideal would be the shortestpossible Replikin permitted by the algorithm which defines a Replikin,that is six amino acids (two ks six to ten amino acids apart), with thehighest possible % k (see deduced Replikin “kkkkhk” (SE) ID NO: 459),which contains 83.3% k, ⅚, and one obligatory “h”). We have therefore,so-to-speak, taken what appears to be, or might be, the nextevolutionary step, not apparently as yet taken by the organismsthemselves, and devised the resultant deduced Replikins to use asgeneral vaccines.

These Replikins which we have deduced have maximum % ‘k’s, thereforemaximum potential binding capacity, plus the constituent ‘h’ bydefinition required for the Replikin, giving the potential for ‘h’connection to redox energy systems. These devised Replikins are leastlikely to be cleaved by organisms because of their short length(proteins are cleaved to 6 to 10 amino acids long in processing forpresentation to and recognition by immune cells), therefore most likelyto present intact to immune-forming apparatuses in the organism to whichthey are administered, and, because of their high k content, they aremost likely to generate a maximum immune response which mimics and mayincrease the maximum such response which can be generated against shorthomologous high mortality Replikins.

Further, we have found that high % k Replikins generate the highestantibody responses when administered to rabbits. These syntheticpeptides, designed by us, are designated as Universal syntheticepitopes, or “UTOPE's”, and the vaccines based upon these UTOPEs, aredesignated “UVAX”s. UVAXs, deduced synthetic vaccines, may be used assole vaccines or as adjuvants when administered with more specificReplikin vaccines or other vaccines. The following are examples ofdeduced UTOPEs and UVAXs:

DEVISED SYNTHETIC REPLIKIN (UTOPE OR UVAX) SEQ ID NO: KKKKHK 459 KKKHKK460 KKHKKK 461 KHKKKK 462 KKKKKKH 463 KKKKKHK 464 KKKKHKK 465 KKKHKKK466 KKHKKKK 467 KHKKKKK 468 HKKKKKK 469

Recognin and/or Replikin peptides may be administered to a subject toinduce the immune system of the subject to produce anti-Replikinantibodies. Generally, a 0.5 to about 2 mg dosage, preferably a 1 mgdosage of each peptide is administered to the subject to induce animmune response. Subsequent dosages may be administered if desired.

The Replikin sequence structure is associated with the function ofreplication. Thus, whether the Replikins of this invention are used fortargeting sequences that contain Replikins for the purpose of diagnosticidentification, promoting replication, or inhibiting or attackingreplication, for example, the structure-function relationship of theReplikin is fundamental.

It is preferable to utilize only the specific Replikin structure whenseeking to induce antibodies that will recognize and attach to theReplikin fragment and thereby cause destruction of the cell. Even thoughthe larger protein sequence may be known in the art as having a“replication associated function,” vaccines using the larger proteinoften have failed or proven ineffective.

Although the present inventors do not wish to be held to a singletheory, the studies herein suggest that the prior art vaccines areineffective because they are based on the use of the larger proteinsequence. The larger protein sequence invariably has one or moreepitopes (independent antigenic sequences that can induce specificantibody formation); Replikin structures usually comprise one of thesepotential epitopes. The presence of other epitopes within the largerprotein may interfere with adequate formation of antibodies to theReplikin, by “flooding” the immune system with irrelevant antigenicstimuli that may preempt the Replikin antigens, See, e.g., Webster, R.G., J. Immunol., 97(2):177-183 (1966); and Webster et al., J. Infect.Dis., 134:48-58, 1976; Klenerman et al, Nature 394:421-422 (1998) for adiscussion of this well-known phenomenon of antigenic primacy wherebythe first peptide epitope presented and recognized by the immune systemsubsequently prevails and antibodies are made to it even though otherpeptide epitopes are presented at the same time. This is another reasonthat, in a vaccine formulation, it is important to present the constantReplikin peptide to the immune system first, before presenting otherepitopes from the organism so that the Replikin is not pre-empted butlodged in immunological memory.

The formation of an antibody to a non-Replikin epitope may allow bindingto the cell, but not necessarily lead to cell destruction. The presenceof structural “decoys” on the C-termini of malaria proteins is anotheraspect of this ability of other epitopes to interfere with binding ofeffective anti-Replikin antibodies, since the decoy epitopes have manylysine residues, but no histidine residues. Thus, decoy epitopes maybind anti-Replikin antibodies, but may keep the antibodies away fromhistidine-bound respiratory enzymes. Treatment may therefore be mostefficacious in two stages: 1) proteases to hydrolyze decoys, then; 2)anti-Replikin antibodies or other anti-Replikin agents.

It is well known in the art that in the course of antibody productionagainst a “foreign” protein, the protein is first hydrolyzed intosmaller fragments. Usually fragments containing from about six to tenamino acids are selected for antibody formation. Thus, if hydrolysis ofa protein does not result in Replikin-containing fragments,anti-Replikin antibodies will not be produced. In this regard, it isinteresting that Replikins contain lysine residues located six to tenamino acids apart, since lysine residues are known to bind to membranes.

Furthermore, Replikin sequences contain at least one histidine residue.Histidine is frequently involved in binding to redox centers. Thus, anantibody that specifically recognizes a Replikin sequence has a betterchance of inactivating or destroying the cell in which the Replikin islocated, as seen with anti-malignin antibody, which is perhaps the mostcytotoxic anti-cancer antibody yet described, being active at picogramsper cell.

One of the reasons that vaccines directed towards a particular proteinantigen of a disease causing agent have not been fully effective inproviding protection against the disease (such as foot and mouth vaccinewhich has been developed against the VP 1 protein or large segments ofthe VP 1 protein) is that the best antibodies have not been produced,that is—it is likely that the antibodies to the Replikins have not beenproduced. Replikins have not been produced. That is, either epitopesother than Replikins present in the larger protein fragments mayinterfere according to the phenomenon of antigenic primacy referred toabove, and/or because the hydrolysis of larger protein sequences intosmaller sequences for processing to produce antibodies results in lossof integrity of any Replikin structure that is present, e.g., theReplikin is cut in two and/or the histidine residue is lost in thehydrolytic processing. The present studies suggest that for an effectivevaccine to be produced, the Replikin sequences, and no other epitope,should be used as the vaccine. For example, a vaccine of the inventioncan be generated using any one of the Replikin peptides identified bythe three-point recognition system.

Particularly preferred peptides—for example—an influenza vaccine includepeptides that have been demonstrated to be conserved over a period ofone or more years, preferably about three years or more, and/or whichare present in a strain of influenza virus shown to have the highestincrease in concentration of Replikins relative to Replikinconcentration in other influenza virus strains, e.g., an emergingstrain. The increase in Replikin concentration preferably occurs over aperiod of at least about six months to one year, preferably at leastabout two years or more, and most preferably about three years or more.Among the preferred Replikin peptides for use in an influenza virusvaccine are those Replikins observed to “re-emerge” after an absencefrom the hemagglutinin amino acid sequence for one or more years.

The Replikin peptides of the invention, alone or in various combinationsare administered to a subject, preferably by i.v. or intramuscularinjection, in order to stimulate the immune system of the subject toproduce antibodies to the peptide. Generally the dosage of peptides isin the range of from about 0.1 μg to about 10 mg, preferably about 10 μgto about 1 mg, and most preferably about 50 μg to about 500 μg. Theskilled practitioner can readily determine the dosage and number ofdosages needed to produce an effective immune response.

Quantitative Measurement Early Response(s) to Replikin Vaccines

The ability to measure quantitatively the early specific antibodyresponse in days or a few weeks to a Replikin vaccine is a majorpractical advantage over other vaccines for which only a clinicalresponse months or years later can be measured.

Adjuvants

Various adjuvants may be used to enhance the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels, such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, key limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG andCorynebacterium parvum. In addition to the use of synthetic UTOPEs asvaccines in themselves, UTOPEs can be used as adjuvants to otherReplikin vaccines and to non-Replikin vaccines.

Replikin Nucleotide Sequences

Replikin DNA or RNA may have a number of uses for the diagnosis ofdiseases resulting from infection with a virus, bacterium or otherReplikin encoding agent. For example, Replikin nucleotide sequences maybe used in hybridization assays of biopsied tissue or blood, e.g.,Southern or Northern analysis, including in situ hybridization assays,to diagnose the presence of a particular organism in a tissue sample oran environmental sample, for example. The present invention alsocontemplates kits containing antibodies specific for particularReplikins that are present in a particular pathogen of interest, orcontaining nucleic acid molecules (sense or antisense) that hybridizespecifically to a particular Replikin, and optionally, various buffersand/or reagents needed for diagnosis.

Also within the scope of the invention are oligoribonucleotidesequences, that include antisense RNA and DNA molecules and ribozymesthat function to inhibit the translation of Replikin- orrecognin-containing mRNA. Both antisense RNA and DNA molecules andribozymes may be prepared by any method known in the art. The antisensemolecules can be incorporated into a wide variety of vectors fordelivery to a subject. The skilled practitioner can readily determinethe best route of delivery, although generally i.v. or i.m. delivery isroutine. The dosage amount is also readily ascertainable.

Particularly preferred antisense nucleic acid molecules are those thatare complementary to a Replikin sequence contained in a mRNA encoding,for example, an influenza virus polypeptide, wherein the Replikinsequence comprises from 7 to about 50 amino acids including (1) at leastone lysine residue located six to ten residues from a second lysineresidue; (2) at least one histidine residue; and (3) at least 6% lysineresidues. More preferred are antisense nucleic acid molecules that arecomplementary to a Replikin present in the coding strand of the gene orto the mRNA encoding the influenza virus hemagglutinin protein, whereinthe antisense nucleic acid molecule is complementary to a nucleotidesequence encoding a Replikin that has been demonstrated to be conservedover a period of six months to one or more years and/or which arepresent in a strain of influenza virus shown to have an increase inconcentration of Replikins relative to Replikin concentration in otherinfluenza virus strains. The increase in Replikin concentrationpreferably occurs over a period of at least six months, preferably aboutone year, most preferably about two or three years or more.

Similarly, antisense nucleic acid molecules that are complementary tomRNA those that are complementary to a mRNA encoding bacterial Replikinscomprising a Replikin sequence of from 7 to about 50 amino acidsincluding (1) at least one lysine residue located six to ten residuesfrom a second lysine residue; (2) at least one histidine residue; and(3) at least 6% lysine residues. More preferred are antisense nucleicacid molecules that are complementary to the coding strand of the geneor to the mRNA encoding a protein of the bacteria.

Further Aspects of Replikins

In an aspect of the present invention a method of preventing or treatinga virus infection comprising administering to a patient in need thereofa preventive or therapeutic virus vaccine is provided comprising atleast one isolated Replikin present in a protein of an emerging strainof the virus and a pharmaceutically acceptable carrier and/or adjuvant.In a further aspect of the invention the isolated or synthesizedpeptides are influenza virus peptides. In yet a further aspect of theinvention, the isolated or synthesized peptides are H5N1 influenza viruspeptides

The present invention also provides a method of making a preventive ortherapeutic virus vaccine comprising:

-   -   (1) identifying a strain of a virus as an emerging strain,    -   (2) selecting at least one Replikin sequence present in the        emerging strain as a peptide template for the virus vaccine        manufacture,    -   (3) synthesizing peptides having the amino acid sequence of the        at least one Replikin sequence selected in step (2), and    -   (4) combining a therapeutically effective amount of the peptides        of step (3) with a pharmaceutically acceptable carrier and/or        adjuvant.        In a further aspect of the method of making a preventive or        therapeutic virus vaccine, the isolated Replikin is from        influenza virus. In still a further aspect, the isolated        Replikin is from an influenza H5N1 virus.

In another aspect, the invention is directed to a method of identifyingan emerging strain of a virus for diagnostic, preventive or therapeuticpurposes comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of the virus;    -   (2) analyzing the amino acid sequence of the at least one        isolate of each strain of the plurality of strains of the virus        for the presence and concentration of Replikin sequences;    -   (3) comparing the concentration of Replikin sequences in the        amino acid sequence of the at least one isolate of each strain        of the plurality of strains of the virus to the concentration of        Replikin sequences observed in the amino acid sequence of each        of the strains from at least one earlier time period to provide        the concentration of Replikins for at least two time periods,        said at least one earlier time period being within about six        months to about three years prior to step (1); and    -   (4) identifying the strain of the virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods.

In another aspect of the invention there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to a Replikin sequence, said process comprisingadministering to the subject an effective amount of a dosage of acomposition comprising at least one Replikin peptide. A further aspectof the present invention comprises at least one peptide that is presentin an emerging strain of the organism if such new strain emerges.Another aspect of the present invention comprises at least one peptidethat is present in influenza H5N1.

The present invention also provides antibodies that bind specifically toa Replikin, as defined herein, as well as antibody cocktails containinga plurality of antibodies that specifically bind to Replikins. Anotheraspect of the present invention provides compositions comprising anantibody or antibodies that specifically bind to a Replikin and apharmaceutically acceptable carrier.

In one aspect of the invention there are provided isolated, or separatedfrom other proteins, recombinant, or synthesized peptides or othermethods containing a viral Replikin sequence.

The present application also provides isolated, or separated fromnucleocapsid proteins, amongst others, recombinant, or synthesizedpeptides or other methods containing a viral Replikin sequence.

In another aspect of the invention there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to a viral Replikin sequence, said process comprisingadministering to the subject an effective amount of a dosage of acomposition comprising at least one Replikin peptide. Another aspect ofthe present invention comprises at least one peptide that is present inan emerging strain of the virus if such new strain emerges.

The present invention also provides antibodies that bind specifically toa viral Replikin, as defined herein, as well as antibody cocktailscontaining a plurality of antibodies that specifically bind to viralReplikins. Another aspect of the present invention provides compositionscomprising an antibody or antibodies that specifically bind to a viralReplikin and a pharmaceutically acceptable carrier.

The present invention also provides therapeutic compositions comprisingone or more of isolated Replikin virus peptides and a pharmaceuticallyacceptable carrier.

In another aspect of the invention there is provided an antisensenucleic acid molecule complementary to a virus Replikin mRNA sequence,said Replikin mRNA sequence denoting from 7 to about 50 amino acidscomprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In yet another aspect of the invention there is provided a method ofsimulating the immune system of a subject to produce antibodies toviruses, said method comprising: administering an effective amount of atleast one virus Replikin peptide.

In another aspect, there is provided a method of selecting a viruspeptide for inclusion in a preventive or therapeutic virus vaccinecomprising:

-   -   (1) obtaining at least on isolate of each strain of a plurality        of strains of said virus;    -   (2) analyzing the amino acid sequence of the at least one        isolate of each strain of the plurality of strains of the virus        for the presence and concentration of Replikin sequences;    -   (3) comparing the concentration of Replikin sequences in the        amino acid sequence of the at least one isolate of each strain        of the plurality of strains of the virus to the concentration of        Replikin sequences observed in the amino acid sequence of each        of the strains at least one earlier time period to provide the        concentration of Replikins for at least two time periods, said        at least one earlier time period being within about six months        to about three years prior to step (1);    -   (4) identifying the strain of the virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods; and    -   (5) selecting at least one Replikin sequence present in the        strain of the virus peptide identified in step (4) as a peptide        for inclusion in the virus vaccine.

In one aspect of the invention there are provided isolated orsynthesized influenza virus peptides comprising a Replikin sequence.

In another aspect of the invention, there is provided a process forstimulating the immune system of a subject to produce antibodies thatbind specifically to an influenza virus Replikin sequence, said processcomprising administering to the subject an effective amount of dosage ofa composition comprising at least one influenza virus Replikin peptide.A further aspect of the present invention comprises at least oneReplikin peptide that is present in an emerging strain of influenzavirus. Yet another aspect of the present invention comprises acomposition comprising at least one influenza H5N1 Replikin peptide.

The present invention also provides antibodies that bind specifically toan influenza virus Replikin, as defined herein, as well as antibodycocktails containing a plurality of antibodies that specifically bind toinfluenza virus Replikins. In another aspect of the present invention,there are provided compositions comprising an antibody or antibodiesthat specifically bind to an influenza Replikin and a pharmaceuticallyacceptable carrier.

The present invention also provides therapeutic compositions comprisingone or more of isolated influenza virus peptides having from 7 to about50 amino acids comprising:

-   -   (1) at least one lysine residue located six to ten residues form        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues, and a pharmaceutical acceptable        carrier.

In another aspect of the invention there is provided an antisensenucleic acid molecule complementary to an influenza virus hemagglutininReplikin mRNA sequence, said Replikin mRNA sequence denoting from 7 toabout 50 amino acids comprising:

-   -   (1) at least one lysine residue located six to ten residues from        a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In yet another aspect of the invention there is provided a method ofsimulating the immune system of a subject to produce antibodies toinfluenza virus comprising administering an effective amount of at leastone influenza virus Replikin peptide having from 7 to about 50 aminoacids comprising:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues.

In another aspect, there is provided a method of selecting an influenzavirus peptide for inclusion in a preventive or therapeutic influenzavirus vaccine comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of influenza virus;    -   (2) analyzing the hemagglutinin amino acid sequence of the at        least one isolate of each strain of the plurality of strains of        influenza virus for the presence and concentration of Replikin        sequences;    -   (3) comparing the concentration of Replikin sequences in the        hemagglutinin amino acid sequence of the at least one isolate of        each strain of the plurality of strains of influenza virus to        the concentration of Replikin sequences observed in the        hemagglutinin amino acid sequence of each of the strains at        least one earlier time period to provide the concentration of        Replikins for at least two time periods, said at least one        earlier time period being within about six months to about three        years prior to step (1);    -   (4) identifying the strain of influenza virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods;    -   (5) selecting at least one Replikin sequence present in the        strain of influenza virus peptide identified in step (4) as a        peptide for inclusion in an influenza virus vaccine.

The present invention also provides a method of making a preventive ortherapeutic influenza virus vaccine comprising:

-   -   (1) identifying a strain of influenza virus as an emerging        strain;    -   (2) selecting at least one Replikin sequence present in the        emerging strain as a peptide template for influenza virus        vaccine manufacture,    -   (3) synthesizing peptides having the amino acid sequence of the        at least one Replikin sequence selected in step (2), and    -   (4) combining a therapeutically effective amount of the peptides        of step (3) with a pharmaceutically acceptable carrier and/or        adjuvant.

In another aspect, the invention is directed to a method of identifyingan emerging strain of influenza virus for diagnostic, preventive ortherapeutic purposes comprising:

-   -   (1) obtaining at least one isolate of each strain of a plurality        of strains of influenza virus;    -   (2) analyzing the hemagglutinin amino acid sequence of the at        least one isolate of each strain of the plurality of strains of        influenza virus for the presence and concentration of Replikin        sequences;    -   (3) comparing the concentration of Replikin sequences in the        hemagglutinin amino acid sequence of the at least one isolate of        each strain of the plurality of strains of influenza virus to        the concentration of Replikin sequences observed in the        hemagglutinin amino acid sequence of each of the strains at        least one earlier time period to provide the concentration of        Replikins for at least two time periods, said at least one        earlier time period being within about six months to about three        years prior to step (1); and    -   (4) identifying the strain of influenza virus having the highest        increase in concentration of Replikin sequences during the at        least two time periods.

In yet another aspect of the invention, there is provided a preventiveor therapeutic influenza virus vaccine comprising at least one isolatedReplikin present in the hemagglutinin protein of an emerging strain ofinfluenza virus and a pharmaceutically acceptable carrier and/oradjuvant.

Also provided by the present invention is a method of preventing ortreating influenza virus infection comprising administering to a patientin need thereof a preventive or therapeutic vaccine comprising at leastone isolated Replikin present in the hemagglutinin protein of anemerging strain of influenza virus and a pharmaceutically acceptablecarrier and/or adjuvant.

Computer Software for Identifying Replikins and Related Structures

Identification of Replikin structures, Replikin Scaffold structures anddegenerate Exoskeleton Scaffold structures may be accomplished with theaid of bioinformatics.

Embodiments of the present invention are directed to a system and methodfor identifying and/or locating complex patterns in an amino acidsequence such as Replikin patterns, Replikin Scaffold structures,Exoskeleton Scaffold structures and other complex patterns in amino acidand nucleic acid sequences. According to an aspect of the presentinvention, techniques are provided to facilitate queries of proteindatabases. For protein descriptions received in response to the queries,aspects of the present invention may include a scan of the receivedprotein descriptions to identify and locate Replikin patterns. Accordingto an aspect of the present invention, a Replikin pattern is a sequenceof from 7 to about 50 amino acids that include the following three (3)characteristics, each of which may be recognized as an aspect of thepresent invention: (1) the sequence has at least one lysine residuelocated six to ten amino acid residues from a second lysine residue; (2)the sequence has at least one histidine residue; and (3) at least 6% ofthe amino acids in the sequence are lysine residues. Another aspect ofthe present invention may identify and/or locate a complex amino acidsequence having specified length constraints, which further includes anycombination of the following characteristics: (1) a first amino acidresidue located more than N positions and less than M positions awayfrom a second amino acid residue; (2) a third amino acid residue locatedanywhere in the sequence; and (3) at least R percent of a fourth aminoacid residue. According to yet another aspect, the present invention maycount occurrences of the identified amino acid sequences and may reportthe counted occurrences, either as raw absolute values or as ratios ofthe number of identified amino acid sequences per N amino acids in theprotein. Still another aspect of the present invention may analyze theevolution of identified amino acid sequence patterns in variants of agiven protein over time, and may also analyze the similarities anddifferences between instances of identified amino acid sequence patternsacross a plurality of different proteins over time. As a result of theanalysis, yet another aspect of the present invention may identifypotential amino acid scaffolding structures that appear to be preservedover time and across different proteins, as component elements of theidentified amino acid sequence patterns mutate and/or evolve.

Embodiments of the present invention will be described with reference tothe accompanying drawings, wherein like parts are designated by likereference numerals throughout, and wherein the leftmost digit of eachreference number refers to the drawing number of the figure in which thereferenced part first appears.

FIG. 17 is a high-level block diagram of a computer system incorporatinga system and method for identifying Replikin patterns in amino acidsequences, in accordance with an aspect of the present invention. Asshown in FIG. 17, computer workstation 610 may be a computer having aprocessor and a memory configured to permit a researcher to searchprotein databases and to scan protein descriptions for selected aminoacid patterns. To accomplish these functions, computer workstation 610may include protein and amino acid research system 630, which mayreceive instructions from a user/researcher to conduct protein searchingand amino acid scanning operations. According to an aspect, protein andamino acid research system 630 may further include amino acid sequencescanner 640 that scans and searches retrieved protein and amino acidsequences for specific patterns of amino acids, including Replikinpatterns. Protein and amino acid research system 630 may communicatewith network interface 620 to obtain protein sequences and amino acidsequences from resources on network 660, which may include the Internet.Alternatively, protein and amino acid research system 630 may obtainprotein sequences and amino acid sequences from a local protein database650. In addition, protein and amino acid research system 630 may obtainprotein sequences and amino acid sequences directly from other inputmeans, such as keyboard input. Protein and amino acid research system630 may also communicate with network interface 620 to transmit resultsto other computers on network 660.

Automated Scanning for Replikin Patterns

Embodiments of the present invention may include a generalized methodand system for identifying complex patterns of amino acids withinproteins. For any protein definition identified or selected by proteinand amino acid research system 630, the user may direct aspects of theinvention to search for a variety of complex patterns of amino acids. Asan example of one pattern of amino acids, the present invention providesa method for identifying nucleotide or amino acid sequences that includea Replikin pattern. FIG. 18 is a simple flow chart illustrating ageneral method for locating a Replikin pattern in a sequence of aminoacids, according to an aspect of the present invention. The method 700may begin after a sequence of amino acids has been obtained. Typically,the sequence of amino acids may be represented by alphabetic charactersaccording to the code supplied in FIG. 12. However, other encodings areenvisioned by the present invention as well.

Referring to FIG. 18, once a sequence of amino acids has been obtained,the sequence is searched for a Replikin pattern (710), which comprises asubsequence (or string) of amino acids that includes the followingcharacteristics:

-   -   (1) the string contains from 7 to about 50 amino acids;    -   (2) the string contains at least one lysine residue located 6 to        10 positions from a second lysine residue;    -   (3) the string contains at least one histidine residue; and    -   (4) the string contains at least 6% lysine residues.

Once a string of amino acids is found to match the Replikin pattern, thestring may be identified or marked (720) accordingly.

A given sequence of amino acids may contain many subsequences or stringsthat match the Replikin pattern. Additionally, Replikin patterns mayoverlap each other. Thus, to locate and identify all possible Replikinpatterns in a sequence of amino acids, method 700 may be invokediteratively for each subsequence of amino acids contained within theoriginal sequence of amino acids.

When method 700 is invoked iteratively to identify and locate allpossible Replikin patterns in an amino acid sequence, an aspect of thepresent invention may count the number of resulting Replikin patterns. AReplikin count may be reported as an absolute number. Additionally,aspects of the invention may also determine a ratio of the number ofReplikins per N amino acids in the sequence. For example, an aspect ofthe present invention may determine that a given protein contains aratio of 6 Replikins for every 100 amino acids. Replikin ratios havebeen shown by laboratory experiment and by epidemiological evidence tocorrelate directly to the rate that a given protein replicates. Rapidreplication of proteins may be an indication of disease. For example,the presence of relatively high ratios of Replikin patterns has beencorrelated to epidemics of influenza. Similarly, an increase in thecount of Replikin patterns observed in a protein over time may also bean indication of future disease caused by the organism from which theprotein was obtained (see, e.g., FIG. 15). Thus, the ability to detectand count Replikin patterns within sequences of amino acids is asignificant advantage of the present invention.

Still referring to FIG. 18, aspects of the present invention may utilizemethod 700 to identify and locate other complex patterns of amino acids,which exhibit characteristics similar to Replikin patterns. That is,although some aspects of the present invention may specify exact valuesfor: (1) distances between amino acids, (2) acceptable lengths ofrecognized amino acid sequences, and (3) the percentage or concentrationof specific amino acids, these exact values may also be expressed asvariables. Thus a researcher may employ an aspect of the presentinvention to identify sequences of amino acids in a protein that havethe following characteristics:

-   -   (1) the sequence contains from rmin to rmax amino acids;    -   (2) the sequence contains at least one lysine residue located        kmin to kmax amino acid residues from a second lysine residue;    -   (3) the sequence contains at least one histidine residue; and    -   (4) the sequence contains at least kpercent lysine residues.

FIG. 19 is a flow chart illustrating a generalized method 800 forlocating a plurality of Replikin-like patterns in a given sequence ofamino acids, according to an aspect of the present invention. The method800 begins by locating a first lysine residue in the given sequence(810). Then, the method 800 may determine whether a second lysineresidue resides within kmin to kmax positions of the first lysineresidue (820). As indicated in FIG. 19, kmin and kmax define the limitson the distance between the first and second lysine residues. For atypical Replikin pattern, kmin will equal 6 and kmax will equal 10.However, these values may be varied by a researcher interested indiscovering other similar patterns.

Once method 800 has identified two lysine residues that are close enoughto each other (820), the method 800 may examine every histidine residuethat resides within rmax positions of both the first and second lysineresidues (830). When method 800 is employed to identify and locatetypical Replikin patterns, rmax will usually be set to equal 50. Forevery histidine residue that resides within rmax positions of the twolysine residues identified in steps (810) and (820), method 800 willconstruct the shortest string of amino acid residues that includes thefirst lysine residue, the second lysine residue, and the identifiedhistidine residue (840). Then, method 800 will determine whether thelength of that shortest string is within the desired range—that is,whether it contains at least rmin amino acid residues and no more thanrmax amino acid residues (850). Finally, if the identified string ofamino acids also contains at least kpercent of lysine residues (860),the string will be identified as matching the desired Replikin-likepattern (870).

Still referring to FIG. 19, it is apparent that method 800 may identifyseveral Replikin-like patterns from a single given amino acid sequence.This may happen because method 800 may examine more than one histidineresidue that resides within rmax positions of the two identified lysineresidues. Each identified histidine residue may, in combination with thetwo lysine residues, match the desired Replikin-like pattern.

One aspect of the method illustrated by FIG. 19 is shown in FIG. 20,which is a source code listing containing a procedure for discoveringall Replikin patterns present in a given sequence of amino acids, inaccordance with an aspect of the present invention. The “match”procedure shown in FIG. 20 is programmed in an interpreted shelllanguage called “Tcl” and recognizes Replikins in a straightforwardfashion. As known in the art, the “Tool Command Language” or Tcl(pronounced “tickle”) is a simple interpreted scripting language thathas its roots in the Unix command shells, but which has additionalcapabilities that are well-suited to network communication, Internetfunctionality and the rapid development of graphical user interfaces.

Alternative methods of recognizing Replikin patterns are also covered bythe teachings of the present invention. For example, the match procedureshown in FIG. 20 could be implemented in other programming languagessuch as Java or C or C++. Additionally, alternative aspects of theReplikin recognizing algorithm may identify the characteristics of aReplikin pattern in any order, and may also traverse component aminoacid sequences and subsequences using recursive techniques, iterativetechniques, parallel processing techniques, divide-and-conquertechniques or any combination thereof.

Protein Search Engine

Returning to FIG. 17, the present invention may include a search engineto access and interact with amino acid and protein databases, eitherlocally or over a network such as the Internet, to retrieve proteindefinitions. For example, protein and amino acid research system 630 mayaccept protein search criteria from a user, and may then access aplurality of on-line amino acid and protein database search engines toretrieve protein definitions that match the supplied search criteria.Protein database search criteria may comprise any text string that mayform a valid search term in any of the on-line protein or amino acidsearch engines. Typically, these search criteria relate to text that maybe found in the printout that describes each specific protein. Forexample, if the user supplied the search criteria “influenza type A,”aspects of the present invention may forward this text string to aplurality of Internet protein and amino acid search engines, each ofwhich may then return any protein descriptions found in their databasesthat contained the terms “influenza type A.” Employing amino acidsequence scanner 640, each of the returned protein descriptions may bescanned for the presence of Replikin patterns.

Additional aspects of the present invention may permit a user to selector de-select a plurality of Internet protein search engines and tocustomize the search criteria and protein retrieval capabilities of thepresent invention for each of the selected on-line protein searchengines. Moreover, aspects of the invention may also permit a user toaccess a local protein database 650 or to supply a specific proteindefinition directly, for example, by supplying a local file namecontaining the protein definition, or by other methods known in the artfor supplying parameters to computer software.

Another aspect of the present invention may include a search engine toaccess and interact with amino acid and protein databases on theInternet to retrieve protein definitions or amino acid sequencedefinitions. After accepting protein or amino acid sequence searchcriteria from a user, the present invention may access a plurality ofamino acid and protein database search engines, through on-line access,to retrieve protein definitions or amino acid sequence definitions thatmatch the supplied search criteria.

Initial existing protein search criteria based on existing definitionsmay comprise any text string that may form a valid search term in any ofthe on-line protein or amino acid search engines. Typically, thesesearch criteria relate to text that may be found in the printout thatdescribes each specific protein. For example, if the user supplied thesearch criteria “influenza type A,” the present invention would forwardthis text string to the plurality of Internet protein and amino acidsearch engines, each of which would then return any protein definitionsin their databases that contained the terms “influenza type A.”

A non-limiting aspect of the present invention comprising a proteinsearch engine entitled “Genome Explorer” is included in Appendix A. TheTcl procedure named “GenomalEnquirer” may control the macro leveloperation of the protein search engine (see “proc GenomalEnquirer{database term additionalCriteria}).” Within the procedureGenomalEnquirer, a series of specific on-line protein search engines maybe accessed and queried using the user-supplied protein search terms andadditional criteria. Additional aspects of the invention may permit auser to select or de-select a plurality of Internet protein searchengines and to customize the search criteria and protein retrievalcapabilities of the present invention for each of the selected on-lineprotein search engines. Moreover, aspects of the invention may alsopermit a user to access local protein databases or to supply a specificprotein definition directly, for example, by supplying a local file namecontaining the protein definition, or by other methods known in the artfor supplying parameters to computer software.

Instructions for running the Genome Explorer are included in Appendix B.Screen snapshots of the Genome Explorer application are included inAppendix C.

Replikin Analysis

Embodiments of the present invention may be employed not only toidentify and locate Replikin patterns in amino acid sequences.Embodiments may also be used to discover and analyze similarities in thestructure of Replikin patterns occurring in different proteins, or toanalyze different Replikin patterns occurring in the same protein overtime. FIG. 21 for example, is a table illustrating a Replikin Scaffoldor “fixed scaffold” structure that was preserved in a “Bird Flu”influenza virus over an 87 year period from 1917 to 2004. Embodiments ofthe present invention may assemble a number of discovered Replikinpatterns in proteins, including Replikin patterns discovered in variantsof the same protein. Along with each Replikin pattern, aspects of thepresent invention may also associate a date when each protein was firstidentified. When directed by a researcher, an aspect of the presentinvention may include sorting and displaying a plurality of selectedReplikin patterns according to content, date or other criteria, in orderto reveal substantially fixed amino acid structures that have beenpreserved in Replikin patterns over time and which may be present indifferent proteins as well as variants of the same protein. Further,when directed by a researcher, an aspect of the invention may employknown methods of pattern analysis to compare a plurality of selectedReplikin patterns in order to identify such fixed amino acid structuresautomatically. As an example, in FIG. 21, the illustrated Replikinpatterns appear to demonstrate—in this case—a relatively fixed scaffoldstructure of (usually) 29 amino acids that begins with a pair of lysineresidues (kk) at the amino terminal, ends with a pair of histidineresidues (hh) at the carboxyl terminal, and contains a lysine residue ineither position 8, 10 or 11. This conservation of scaffold structureover decades permits synthetic vaccines to be prepared rapidly andinexpensively. To synthesize such vaccines after a Replikin scaffoldingstructure has been identified, a researcher may select elements of thatscaffolding structure that are conserved over time and which are alsopresent in a current variant of a protein. A vaccine may then beprepared based on the selected elements from the scaffolding structure.Because such vaccines are based on conserved scaffolding structures,they may be effective for multiple years and may also be developed wellin advance of an anticipated outbreak.

The discovery of Replikins themselves, as well as aspects of the presentinvention for identifying and locating Replikin patterns, providestargets for the identification of pathogens, as well as facilitates thedevelopment of anti-pathogen therapies, including vaccines. In general,knowledge of and identification of the Replikin family of peptidesenables development of effective therapies and vaccines for any organismthat harbors Replikins. Specifically, identification of Replikinsprovides for the detection of viruses and virus vaccine development,including the influenza virus. Further, identification of Replikins alsoprovides for the detection of other pathogens, such as malaria, anthraxand small pox virus, in addition to enabling the development oftherapies and vaccines that target Replikin structures. Additionalexamples provided by the identification of Replikins include thedetection of infectious disease Replikins, cancer immune Replikins andstructural protein Replikins.

Embodiments of the present invention enable important Replikin patternsof amino acids to be recognized, located and analyzed in manners thatare not found in the prior art. Using prior art capabilities,researchers have been limited in by existing techniques for describingsequences of amino acids. Indeed, limitations of the prior art have insome ways dampened research in this field, since heretofore it has notbeen possible to specify sequences of amino acids that comprisenon-linear attributes. Until the development of the methods and aspectsof the present invention, descriptions of amino acid sequences werelimited to linear sequences containing, at most, repetitive substringsand logical constraints on substring content. Embodiments of the presentinvention enable a new class of amino acid sequences to be discovered,located and analyzed using tools not found in the prior art. This newclass of amino acids is characterized by attributes such as specificamino acid concentration and distance relationships between specificamino acids. These attributes transcend simple contiguous ordering andthus are not easily described, discovered or located by existing methodsknown in the art.

For example, rather than examining strict amino acid sequence matches(homologies) as is done by other widely used programs such as BLAST, thepresent inventors have discovered a unique quantitative “language”related to rapid replication which defines a new class of amino acidgrouping. Novel computer programs described herein detect instances ofthis new language.

These programs include functionality to search electronic data for aminoacid sub-sequences meeting predetermined criteria. The data, which maybe obtained online, may include data defining a specified group ofprotein sequences. The criteria may include:

i) the occurrence within a protein sequence of two amino acids, in thiscase Lysine(K) and histidine(H) in specific concentrations in thesequence

ii) the spacing of one of these (K) to a second K in the sequence, and

iii) the concentration of one or more amino acids (e.g. K) in apercentage greater than a defined percentage.

Amino acid sequences meeting the above criteria relate to a particularbiological function such as rapid replication.

The programs include the capability to identify Replikin sub-sequencesin genome sequences. One source of the genome sequences may be publishedgenome sequences obtained from online, electronic databases, usingsearch criteria provided by a user. In aspects of the invention, thedatabases may be NCBI (National Center for Biotechnology, Information)or LANL (Los Alamos National Laboratory) databases. The programs furtherinclude the capability to search for arbitrary sub-sequences (i.e., notonly Replikin sub-sequences), based on user-supplied criteria.

In one aspect, a program herein entitled “Genome Explorer” may generatea user interface to prompt a user for search terms. Genome Explorer mayapply the search terms to online databases, such as NCBI or LANLdatabases, to obtain raw sequence data. Additional data may be furtherobtained, such as article names, protein source, strain, serotype andyear of discovery for all the raw sequences which match the searchterms. Once the raw data has been acquired, Genome Explorer may furtherapply additional search criteria to identify Replikin sub-sequenceswithin the raw sequences. The search criteria can be specified by theuser in such a way as to implement relatively strict, or relativelyrelaxed definitions of what can be included in the set of matchingsub-sequences to be reported by Genome Explorer. As it identifiesReplikin sub-sequences, Genome Explorer may compile ongoing statisticsand display a progress bar in a user interface. When Genome Explorercompletes its processing, it may save resulting statistics in a datafile. For example, the data file may be an HTML file that can be openedin any word processor for inspection of results.

In another aspect, a program herein entitled “Dr. Peptide,” searchcriteria may be applied to identify sub-sequences other than Replikinsub-sequences. With Dr. Peptide it is possible to search for, e.g., allinstances of the sequence hlk . . . hlk (SEQ ID NO: 470), separated bynot more than 15 amino acids, in publicly available genome databases.Such searches allow the creation of new statistical profiles and newgroupings of proteins based on meeting these criteria. Dr. Peptide mayinclude much the same functionality as Genome Explorer. For example,like Genome Explorer, Dr. Peptide may, via a user interface, prompt auser for search terms and apply the search terms to online databases,such as NCBI or LANL databases, to obtain raw sequence data. Additionaldata may be further obtained, such as article names, protein source,strain, serotype and year of discovery for all the raw sequences whichmatch the search terms. Once the raw data has been acquired, Dr. Peptidemay further process the data to identify arbitrary sub-sequences andpresent its output in a data file, for example in the form of HTML pagesthat can be opened in any word processor.

Below is a description of one example of a logic sequence that could beincluded in the Genome Explorer program. In the description, an “initialserver inquiry” refers to search criteria to be applied to one or morenetwork elements, such as server computers, storing electronic datarepresenting protein sequences. The network elements may be included inprivate networks or, for example, the Internet. The data may be in theform of a “protein page,” i.e., a quantum of data representing proteinsequences. The character “k” represents a lysine amino acid, and thecharacter “h” represents a histidine amino acid.

Genome Explorer Logic Sequence

Initialize user interface procedures and input fields for searchparameters.

Construct user interface.

wait for user to specify search parameters.

Search parameters include:

(1) words or phrases to be matched in the initial server inquiry toobtain summaries and protein pages,

(2) The allowed distance between k's, expressed as range kmin . . . kmaxfor a sub-sequence to qualify for a set.

(3) The allowed range of distances between an h and the farthest k,expressed as kmin+1 . . . hmax, for a sub-sequence to qualify for theset.

(4) The allowed fraction of k's in the sub-sequence, expressed as xpercent or larger, for the sub-sequence to qualify for the set.

Once search parameters are specified,

Initialize output files in HTML format—these will be used to displayreports.

Compare specified search parameters with previous search.

If the search parameters are identical, reuse cached protein pages asdata input.

If the search parameters are not identical (cached protein pages are notrelevant),

Send the inquiry to the server (NCBI or LANL).

If it did not return all summaries,

Re-send the inquiry requesting all summaries.

For each summary,

Fetch and save the protein page retrieved.

For each protein page retrieved,

If from NCBI,

Parse ASN page.

Extract found sequence data (seq-data.ncbieaa).

Extract article names (descr.*.article.title.*.name).

Extract protein source (source.org.taxname).

Extract strain (subtype).

Derive year discovered.

Derive serotype.

If from LANL,

Parse HTML page for strain, definition, source, year, serotype, and rawnucleotide sequence.

Convert nucleotides to amino acids

by mapping every three nucleotides in sequence

to the corresponding amino acid.

Save parsed value for this protein.

For each parsed page, update user interface as to progress via progressbar, and:

For each sequence data found on the page,

Scan the amino acid sequence data for each sub-sequence matching

(a) The distance between k's is in the range kmin . . . kmax as definedin parameter (2) from the user interface above.

(b) The distance between an h and the farthest k is in the range kmin+1. . . hmax as defined in parameter (3) from the user interface above.

(c) The fraction of k units in the sub-sequence, expressed as x percentor larger as defined in parameter (4) from the user interface above.

and save the range of each matching sub-sequence, including overlaps.

Ignore sequences with no matches.

Accept the sequence with the most sub-sequence matches.

If a sequence was accepted,

Catalog each sequence by the year it was discovered.

For each additional set of criteria,

Check the additional criteria against other parsed fields.

If does not match, do not accept the page.

If the page was accepted,

Add it as a passed page.

Create an HTML page showing the full sequence and all matchedsub-sequences.

If the page was not accepted,

Add it as a failed page.

For each unique matched replikin sequence,

Create an amino acid history HTML page,

Show every protein it occurs in ordered by year.

Create a statistics HTML page displaying the following:

For each year,

Show number of matched proteins and replikin sub-sequences.

Update user interface to reflect that the operation is complete;

Re-initialize input fields to allow next set of search parameters to bespecified by user.

In view of the foregoing description, it may be understood that GenomeExplorer implements a method including applying a plurality of criteriato data representing protein sequences, and based on the criteria,identifying a sub-sequence within the protein sequences, the identifiedsub-sequence having a predetermined allowed range of distance betweenLysine amino acids thereof, and a predetermined allowed range ofdistance between a histidine amino acid and a farthest Lysine acidthereof. An identified sub-sequence may be output to a data file.

The functionality of the herein aspects may be provided on variouscomputer platforms executing program instructions. One such platform1100 is illustrated in the simplified block diagram of FIG. 22. There,the platform 1100 is shown as being populated by a processor 1160, whichcommunicates with a number of peripheral devices via a bus subsystem1150. These peripheral devices typically include a memory subsystem1110, a network interface subsystem 1170, and an input/output (I/O) unit1180. The processor 1160 may be any of a plurality of conventionalprocessing systems, including microprocessors, digital signal processorsand field programmable logic arrays. In some applications, it may beadvantageous to provide multiple processors (not shown) in the platform1100. The processor(s) 1160 execute program instructions stored in thememory subsystem 1110. The memory subsystem 1110 may include anycombination of conventional memory circuits, including electrical,magnetic or optical memory systems. As shown in FIG. 22, the memorysystem may include read only memories 1120, random access memories 1130and bulk storage 1140. Memory subsystem 1110 not only stores programinstructions representing the various methods described herein but alsomay store the data items on which these methods operate. Networkinterface subsystem 1170 may provide an interface to outside networks,including an interface to communications network 1190 comprising, forexample, the Internet. I/O unit 1180 would permit communication withexternal devices, which are not shown.

Several aspects of the present invention are specifically illustratedand described herein. However, it will be appreciated that modificationsand variations of the present invention are covered by the teachings ofthe present invention without departing from the spirit and intendedscope of the invention. Additionally, the teachings of the presentinvention may be adaptable to other sequence-recognizing problems thathave heretofore been addressed using sequential linear analyses limitedto the identification of specific sequences of component elements.

Using the exemplary software contained in Appendix A, the inventors havediscovered in a non-limiting aspect in accordance with the presentinvention that the nucleocapsid protein of the shrimp white spot virushas an exceptionally high Replikin Count as compared to all otherviruses and organisms surveyed for replikins up to the present time(with the exception of malaria). While Replikins have been shown to beessential accompaniments of rapid replication in fungi, yeast, viruses,bacteria, algae, and cancer cells, the inventors have provided the firstdemonstration of the presence of replikins in marine organisms otherthan algae. And, as with algae, the presence of replikins is againrelated to rapid infestations. In shrimp, the white spot virus hasdestroyed millions of dollars of harvest of shrimp, first in easterncountries, and now in western hemisphere countries. At present, there isno effective prevention or treatment. Other examples of Replikin highmortality marine viral disease have been demonstrated by us in fish suchas carp and hemorrhagic disease in salmon, and are probably widespreadin marine ecology and disease.

The presence of repeat sequences of the Replikins of the nucleocapsidprotein of shrimp white spot syndrome virus (WSSV) accounts for theunusually high Replikin Count of 103.8. This virus Replikin Count ismuch higher than the Replikin Counts of for example influenza viruseswhich usually range from less than 1 up to 5 or 7, and is comparableonly to the record Replikin Count (so far) observed in PlasmodiumFalciparum (malaria) of 111. Interestingly, while the shrimp white spotsyndrome organism is a virus, and the Pl. Falciparum is a trypanosome,both spend an essential part of their reproductive cycles in red bloodcells, an unusual host cell whether in shrimp (white spot virus) or man(malaria), both are fulminating rapidly replicating diseases with highmortality rates of their hosts, and both appear to use the same methodsof increasing their high Replikin Counts to such record highs, namely,Replikin Repeats and Replikin Overlap.

As illustrated in Table 10, examples of Replikin Repeats and ReplikinOverlap were found by the applicants in the above nucleocapsid proteinof the shrimp white spot syndrome virus as seen below. 497 Replikinswere discovered in the white spot virus using the exemplary softwareprovided in Appendix A. Of those 497, the replikins illustrated below inTable 10 were selected for their short sequences and high concentrationof lysine which, as demonstrated throughout this application, appears tobe associated with high mortality. The chosen sequences are easier andless expensive to synthesize than the longer sequences that are notincluded in Table 10.

Table 10 illustrates intramolecular Replikin Repeats and ReplikinOverlap in shrimp white spot syndrome virus (WSSV) nucleocapsid protein(VP35) gene with a Replikin Count (number of replikins per 100 aminoacids) of 103.8 (497 total replikins per 479 amino acids).

TABLE 10Intramolecular Replikin Repeats and Replikin Overlap in shrimp white spotsyndrome virus (WSSV) nucleocapsid protein (VP35) gene withReplikin Count = Number of Replikins per 100 amino acids = 497/479 =103.8 and with thymidine kinase and thymidylate kinase activity.Individual Replikins at Different Positions Replikinin the same Molecule, in order of appearance ID Number in the sequence(to be assigned)                            k24 q25 l26 l27 h28 l29 k30k30 v31 h32 l33 d34 v35 k36k30 v31 h32 l33 d34 v35 k36 g37 v38 k39 q40 l41 l42 h43                                k39 q40 l41 l42 h43 l44 k45k66 k67 n68 v69 k70 s71 a72  k73 q74  l 75 p76 h77              k70 s71 a72 k73 q74 l75 p76 h77 l78 k79k160 k161 n162 v163 k164 s165 a166  k167 g168 l169 p170 h171k239 k240 n241 v242 k243 s244 a245  k246 q247 l248 p249 h250k303 k304 n305 v306 k307 s308 a309  k310 q311 l312 p313 h314k397 k398 n399 v400 k401 s402 a403  k404 q405 l406 p407 h408 *Note inthe shrimp virus the repeated use of identical whole Replikin sequences(underlined) and partial Replikin sequences (italicized) in differentpositions in the one molecule (each amino acid is numbered according toits order in the sequence).

Now that we have been able to identify these Replikins using thesoftware described in this application, we can synthesize each of themand use them as targets for antibody and other inhibitory products andfor specific synthetic vaccines against the shrimp white spot syndromevirus, specifically directed against each repeating Replikin.

The phenomenon of repeats is well known in protein structure. What isunique and specific in this case is that these are Replikin repeats.Thus while repeat of a specific Replikin sequence increases the ReplikinCount within a specific molecule and is associated with more rapidreplication as in the case of ATPase in Pl. Falciparum in malaria, thushas apparent survival value for the molecule and the organism whichcontains it, at the same time it provides an increasing vulnerability,an ‘Achilles Heel’ so to speak. Thus the Replikin Repeat provides ahigher concentration per molecule, additional target sites for attack byspecific antibodies as generated by specific synthetic vaccines producedagainst these Replikins and other specific anti-Replikin agents. Thesenew targets were previously unavailable because they could not beidentified.

Complex Amino Acid Analysis

A further aspect of the present invention comprises a protein searchengine directed to recognizing generalized amino acid and nucleic acidpatterns on-line databases. Appendix D is an exemplary protein searchengine directed to recognizing complex amino acid patterns such asScaffold Exoskeletons. Appendix D is entitled “Dr. Peptide.” Appendix Dis an exemplary non-limiting aspect of the present invention and isdesigned to recognize generalized amino acid patterns in addition to theReplikin pattern.

Below is a description of one example of a logic sequence that could beincluded in the Dr. Peptide program. In the description, an “initialserver inquiry” refers to search criteria to be applied to one or morenetwork elements, such as server computers, storing electronic datarepresenting protein sequences. The network elements may be included inprivate networks or, for example, the Internet. The data may be in theform of a “protein page,” i.e., a quantum of data representing proteinsequences.

Dr. Peptide Logic Sequence

Initialize user interface procedures and input fields for searchparameters.

Construct user interface.

wait for user to specify search parameters, including:

(1) words or phrases to be matched in the initial server inquiry toobtain summaries and protein pages,

(2) a set of specific amino acids which must be included in anysub-sequences qualifying for a set.

(3) a set of specific amino acids which must be excluded from anysub-sequences qualifying for the set.

(4) minimum m and maximum n sizes for the permissible size spacing gapwhich is to be applied to the set inclusion and exclusion criteria (2)and (3).

Once search parameters are specified,

Query:

If the saved protein pages are not relevant,

Send the inquiry to the server (NCBI or LANL).

If it did not return all summaries,

Re-send the inquiry requesting all summaries.

For each summary,

Fetch and save the protein page.

For each protein page,

If from NCBI,

Parse ASN page.

Extract found sequence data (seq-data.ncbieaa).

Extract article names (descr.*.article.title.*.name).

Extract protein source (source.org.taxname).

Extract strain (subtype).

Derive year discovered.

Derive serotype.

If from LANL,

Parse HTML page for strain, definition, source, year,

serotype, and raw nucleotide sequence.

Convert nucleotides to amino acids

by mapping every three nucleotides in sequence

to the corresponding amino acid.

Save parsed value for this protein.

For each parsed page,

For each sequence data found on the page,

Scan the amino acid sequence data for each sub-sequence matching.

The match patterns are a sequence of alternative steps:

(a) An amino acid in the amino acid sequence data is in a set ofspecific amino acids as defined in user parameter (2) above.

(b) An amino acid in the amino acid sequence data is not in the set ofspecific amino acids defined in user parameter (3) above.

(c) An amino acid in the amino acid sequence data has a spacing gap of mto n amino acids from another amino acid in the amino acid sequence dataas defined in user parameter (4) above.

The initial sub-sequence set is all possible terminal sequences, or“tails” of the sequence data at the first pattern step,

While the set of sub-sequences is not empty,

Remove one sub-sequence and record how far in the pattern string itsevaluation has reached.

If the amino acid at the current pattern step

Is in a set of specific amino acids,

If the next amino acid of the sub-sequence is also in the set of aminoacids,

Add the elongated sub-sequence and next pattern step

to the sub-sequence set.

Is not in a set of specific amino acids.

If the next amino acid of the sub-sequence is not one of the set ofamino acids,

Add the elongated sub-sequences and next pattern step

to the sub-sequence set.

Has a gap of m to n any amino acids.

First, elongate each sub-sequence for each possible length m through n

Then add each elongated version of the sub-sequence to the sub-sequenceset

If the above pattern is exhausted,

The sub-sequence is a matched sub-sequence.

Ignore sequences with no matches.

Accept the sequence with the most matches.

If a sequence has been accepted,

Catalog each sub-sequence by the year it was discovered.

For each additional criteria,

Check the additional criteria against other parsed fields.

If it does not match, do not accept the page.

If the page was accepted,

Add it as a passed page.

Create an HTML page showing the full sequence and all matchedsubsequences.

If the page was not accepted,

Add it as a failed page.

In view of the foregoing description, it may be understood that Dr.Peptide implements a method including applying a plurality of criteriato data representing protein sequences, and based on the criteria,identifying arbitrary sub-sequences within the protein sequences. Anidentified sub-sequence may be output to a data file. The criteria mayinclude:

a set {a} of amino acids to be included in the sub-sequence;

a set of amino acids to be excluded from the sub-sequence; and

a minimum and a maximum permissible gap between members of sets {a} and{b}.

A non-limiting and exemplary aspect of the invention employs the complexamino acid analysis aspect of the invention to analyze Replikin Scaffoldsequences in earlier strains of influenza that have degenerated intonon-replikin sequences but maintained the scaffold structure of theReplikin Scaffold. As an example of the use of the exemplary andnon-limiting software program in Appendix D to recognize generalizedamino acid patterns, the inventors first discovered by visual scanningof protein sequences (now by Dr. Peptide software) that what was inearlier-arising specimens of a particular influenza species a ReplikinScaffold, was in later specimens changed as follows:

-   -   1) The length of 29 amino acids was preserved;    -   2) The first two amino acid positions (1 and 2) were preserved,        i.e. KK;    -   3) The last two amino acid positions (28 and 29) were preserved,        i.e. HH;    -   4) But there was no longer a K which was 6 to 10 amino acids        from KK (needed for the definition of a Replikin).

Thus this Scaffold is no longer a Replikin Scaffold, but now is aScaffold Exoskeleton so to speak. While Replikin Scaffolds areassociated with high Replikin Counts and the occurrence of epidemics,Scaffold Exoskeletons are associated with virus dormancy and thereduction or end of the epidemic. Thus Scaffold Exoskeletons appear tobe degenerative structures left as residues when Replikin Scaffolds andspecific viral outbreaks are declining, thus a useful diagnosticstructure for this purpose. This confirms the revelation and use ofReplikin Scaffolds as 1) targets for anti-rapid replication agents suchas antibodies or small inhibitory RNAs and 2) the basis of anti-viralvaccines. Software according to aspects of the present invention maycomprise logic to obtain and analyze protein sequences to identifysequences having characteristics 1, 2, 3 and 4 above. For example,Scaffold Exoskeletons can now be detected and counted in any proteinsequence by the exemplary software in Appendix D.

Another non-limiting aspect in accordance with the present invention isa method of identifying a Replikin Scaffold comprising identifying aseries of peptides comprising about 17 to about 30 amino acids andfurther comprising

-   -   (1) identifying a terminal lysine;    -   (2) identifying a terminal histidine and another histidine in        the residue potion immediately adjacent to the terminal        histidine;    -   (3) identifying at least one lysine within about 6 to about 10        amino acid residues from at least one other lysine; and    -   (4) identifying at least about 6% lysines.

In a non-limiting aspect in accordance with the present invention themethod of identifying a Replikin Scaffold may comprise identifying asingle or plurality of individual members of the series of a ReplikinScaffold.

In a preferred non-limiting aspect in accordance with the presentinvention the method of identifying a Replikin Scaffold furthercomprises the identification of a second lysine immediately adjacent tothe terminal lysine. Software according to aspects of the presentinvention may comprise logic to obtain and analyze protein sequences toidentify sequences using steps 1, 2, 3 and 4 above.

The Tcl Programming Language

Tcl (the “Tool Command Language,” pronounced “tickle”) is a simpleinterpreted scripting language that has its roots in the Unix commandshells, but which has additional capabilities that are well-suited tonetwork communication, Internet functionality and the rapid developmentof graphical user interfaces. Tcl was created by John K. Ousterhout atthe University of California at Berkeley in 1988. Originally conceivedas a reusable, embeddable language core for various software tools, itis now widely used in applications including web scripting, testautomation, network and system management, and in a variety of otherfields.

In aspects, Genome Explorer and Dr. Peptide may be coded in Tcl/Tk, ascripting programming language that includes powerful facilities forinternet access, user interface design, and string manipulation. BecauseTcl/Tk has been ported to nearly all available computer architecturesand is familiar to those skilled in the art, programs written in Tcl/Tkcan be run on nearly any operating system. Source code for specificimplementations of Genome Explorer and Dr. Peptide are provided inAppendices A and D. The specific implementations are provided by way ofillustration and example only, and the present invention is not in anyway limited to the specific implementations illustrated.

Other Uses of the Three Point Recognition Method

Since “3-point-recognition” is a proteomic method that specifies aparticular class of proteins, using three or more different recognitionpoints for other peptides similarly should provide useful informationconcerning other protein classes. Further, the “3-point-recognition”method is applicable to other recognins, for example to the TOLL‘innate’ recognition of lipopolyssacharides of organisms. The threepoint recognition method may also be modified to identify other usefulcompounds of covalently linked organic molecules, including othercovalently linked amino acids, nucleotides, carbohydrates, lipids orcombinations thereof. In this aspect of the invention a sequence isscreened for subsequences containing three or more desired structuralcharacteristics. In the case of screening compounds composed ofcovalently linked amino acids, lipids or carbohydrates the subsequenceof 7 to about 50 covalently linked units should contain (1) at least onefirst amino acid, carbohydrate or lipid residue located seven to tenresidues from a second of the first amino acid, carbohydrate or lipidresidue; (2) encoding at least one second amino acid, lipid orcarbohydrate residue; and (3) at least 6% of the first amino acid,carbohydrate or lipid residue. In the case of screening nucleotidesequences, the subsequence of about 21 to about 150 nucleotides shouldcontain (1) at least one codon encoding a first amino acid locatedwithin eighteen to thirty nucleotides from a second codon encoding thefirst amino acid residue; (2) at least one second amino acid residue;and (3) encodes at least 6% of said first amino acid residue.

Several aspects of the present invention are specifically illustratedand described herein. However, it will be appreciated that modificationsand variations of the present invention are encompassed by the aboveteachings and within the purview of the appended claims withoutdeparting from the spirit and intended scope of the invention.

Example 1 Process for Extraction, Isolation and Identification ofReplikins and the Use of Replikins to Target, Label or DestroyReplikin-Containing Organisms

a) Algae

The following algae were collected from Bermuda water sites and eitherextracted on the same day or frozen at −20 degrees C. and extracted thenext day. The algae were homogenized in a cold room (at 0 to 5 degreesC) in 1 gram aliquots in neutral buffer, for example 100 cc. of 0.005Mphosphate buffer solution, pH 7 (“phosphate buffer”) for 15 minutes in aWaring blender, centrifuged at 3000 rpm, and the supernatantconcentrated by perevaporation and dialyzed against phosphate buffer inthe cold to produce a volume of approximately 15 ml. The volume of thisextract solution was noted and an aliquot taken for protein analysis,and the remainder was fractionated to obtain the protein fraction havinga pK range between 1 and 4.

The preferred method of fractionation is chromatography as follows: Theextract solution is fractionated in the cold room (4° C.) on a DEAEcellulose (Cellex-D) column 2.5×11.0 cm, which has been equilibratedwith 0.005M phosphate buffer. Stepwise eluting solvent changes are madewith the following solutions:

-   -   Solution 1—4.04 g. NaH2P04 and 0.5 g NaH2P04 are dissolved in 15        litres of distilled water (0.005 molar, pH 7);    -   Solution 2—8.57 g. NaH2P04 is dissolved in 2,480 ml. of        distilled water;    -   Solution 3—17.1 g. of NaH2P04 is dissolved in 2480 ml of        distilled water (0.05 molar, pH 4.7);    -   Solution 4—59.65 g. of NaH2P04 is dissolved in 2470 ml distilled        water (0.175 molar);    -   Solution 5—101.6 g. of NaH2P04 is dissolved in 2455 ml distilled        water (pH 4.3);    -   Solution 6—340.2 g. of NaH2P04 is dissolved in 2465 of distilled        water (1.0 molar, pX-i 4.1);    -   Solution 7—283.63 g. of 80% phosphoric acid (H3P04) is made up        in 2460 ml of distilled water (1.0 molar, pH 1.0).

The extract solution, in 6 to 10 ml volume, is passed onto the columnand overlayed with Solution 1, and a reservoir of 300 ml of Solution 1is attached and allowed to drip by gravity onto the column. Three mlaliquots of eluant are collected and analyzed for protein content at OD280 until all of the protein to be removed with Solution 1 has beenremoved from the column. Solution 2 is then applied to the column,followed in succession by Solutions 3, 4, 5, 6 aid 7 until all of theprotein which can, be removed with each Solution is removed from thecolumn. The eluates from Solution 7 are combined, dialyzed againstphosphate buffer, the protein content determined of both dialysand anddialyzate, and both analyzed by gel electrophoresis. One or two bands ofpeptide or protein of molecular weight between 3,000 and 25,000 Daltonsare obtained in Solution 7. For example the algae Caulerpa mexicana,Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpaverticillata, Halimeda tuna, and Penicillos capitatus, after extractionand treatment as above, all demonstrated in Solution 7 eluates sharppeptide bands in this molecular weight region with no contaminants.These Solution 7 proteins or their eluted bands are hydrolyzed, and theamino acid composition determined. The peptides so obtained, which havea lysine composition of 6% or greater are Replikin precursors. TheseReplikin peptide precursors are then determined for amino acid sequenceand the Replikins are determined by hydrolysis and mass spectrometry asdetailed in U.S. Pat. No. 6,242,578 B1. Those that fulfill the criteriadefined by the “3-point-recognition” method are identified as Replikins.This procedure can also be applied to obtain yeast, bacterial and anyplant Replikins.

b) Virus

Using the same extraction and column chromatography separation methodsas above in a) for algae, Replikins in virus-infected cells are isolatedand identified.

c) Tumor Cells In Vivo and In Vitro Tissue Culture

Using the same extraction and column chromatography separation methodsas above in a) for algae, Replikins in tumor cells are isolated andidentified. For example, Replikin precursors of Astrocytin isolated frommalignant brain tumors, Malignin (Aglyco 1OB) isolated from glioblastomatumor cells in tissue culture, MCF7 mammary carcinoma cells in tissueculture, and P3J Lymphoma cells in tissue culture each treated as abovein a) yielded Replikin precursors with lysine content of 9.1%, 6.7%,6.7%, and 6.5% respectively. Hydrolysis and mass spectrometry of Aglyco1OB as described in Example 10 U.S. Pat. No. 6,242,578 B1 produced theamino acid sequence, ykagvaflhkkndiide (SEQ ID NO: 471) the 16-merReplikin.

Example 2

As an example of diagnostic use of Replikins: Aglyco 1OB or the 16-merReplikin may be used as antigen to capture and quantify the amount ofits corresponding antibody present in serum for diagnostic purposes areas shown in FIGS. 2, 3, 4 and 7 of U.S. Pat. No. 6,242,578 B1.

As an example of the production of agents to attach to Replikins forlabeling, nutritional or destructive purposes: Injection of the 16-merReplikin into rabbits to produce the specific antibody to the 16-merReplikin is shown in Example 6 and FIGS. 9A and 9B of U.S. Pat. No.6,242,578 B1.

As an example of the use of agents to label Replikins: The use ofantibodies to the 16-mer Replikin to label specific cells which containthis Replikin is shown in FIG. 5 and Example 6 of U.S. Pat. No.6,242,578 B1.

As an example of the use of agents to destroy Replikins: The use ofantibodies to the 16-mer Replikin to inhibit or destroy specific cellswhich contain this Replikin is shown in FIG. 6 of U.S. Pat. No.6,242,578 B1.

Example 3

Analysis of sequence data of isolates of influenza virus hemagglutininprotein or neuraminidase protein for the presence and concentration ofReplikins is carried out by visual scanning of sequences or through useof a computer program based on the 3-point recognition system describedherein. Isolates of influenza virus are obtained and the amino acidsequence of the influenza hemagglutinin and/or neuraminidase protein isobtained by any art known method, such as by sequencing thehemagglutinin or neuraminidase gene and deriving the protein sequencetherefrom. Sequences are scanned for the presence of new Replikins,conservation of Replikins over time and concentration of Replikins ineach isolate. Comparison of the Replikin sequences and concentrations tothe amino acid sequences obtained from isolates at an earlier time, suchas about six months to about three years earlier, provides data that areused to predict the emergence of strains that are most likely to be thecause of influenza in upcoming flu seasons, and that form the basis forseasonal influenza peptide vaccines or nucleic acid based vaccines.Observation of an increase in concentration, particularly a stepwiseincrease in concentration of Replikins in a given strain of influenzavirus for a period of about six months to about three years or more is apredictor of emergence of the strain as a likely cause of influenzaepidemic or pandemic in the future.

Peptide vaccines or nucleic acid-based vaccines based on the Replikinsobserved in the emerging strain are generated. An emerging strain isidentified as the strain of influenza virus having the highest increasein concentration of Replikin sequences within the hemagglutinin and/orneuraminidase sequence during the time period. Preferably, the peptideor nucleic acid vaccine is based on or includes any Replikin sequencesthat are observed to be conserved in the emerging strain. ConservedReplikins are preferably those Replikin sequences that are present inthe hemagglutinin or neuraminidase protein sequence for about two yearsand preferably longer. The vaccines may include any combination ofReplikin sequences identified in the emerging strain.

For vaccine production, the Replikin peptide or peptides identified asuseful for an effective vaccine are synthesized by any method, includingchemical synthesis and molecular biology techniques, including cloning,expression in a host cell and purification therefrom. The peptides arepreferably admixed with a pharmaceutically acceptable carrier in anamount determined to induce a therapeutic antibody reaction thereto.Generally, the dosage is about 0.1 mg to about 10 mg.

The influenza vaccine is preferably administered to a patient in needthereof prior to the onset of “flu season.” Influenza flu seasongenerally occurs in late October and lasts through late April. However,the vaccine may be administered at any time during the year. Preferably,the influenza vaccine is administered once yearly, and is based onReplikin sequences observed to be present, and preferably conserved inthe emerging strain of influenza virus. Another preferred Replikin forinclusion in an influenza vaccine is a Replikin demonstrated to havere-emerged in a strain of influenza after an absence of one or moreyears.

Example 4

Analysis of sequence data of isolates of coronavirus nucleocapsid, orspike, or envelope, or other protein for the presence and concentrationof Replikins is carried out by visual scanning of sequences or throughuse of a computer program based on the 3-point recognition methoddescribed herein. Isolates of coronavirus are obtained and the aminoacid sequence of the coronavirus protein is obtained by any method knownin the art, such as by sequencing the protein's gene and deriving theprotein sequence therefrom. Sequences are scanned for the presence ofnew Replikins, conservation of Replikins over time and concentration ofReplikins in each isolate. Comparison of the Replikin sequences andconcentrations to the amino acid sequences obtained from isolates at anearlier time, such as about six months to about three years earlier,provides data that are used to predict the emergence of strains that aremost likely to be the cause an outbreak or pandemic, and that form thebasis for coronavirus peptide vaccines or nucleic acid based vaccines.Observation of an increase in concentration, particularly a stepwiseincrease in concentration of Replikins in a given class, or strain, ofcoronavirus for a period of about six months to about three years ormore is a predictor of emergence of the strain as a likely cause of anepidemic or pandemic, such as SARS, in the future.

Peptide vaccines or nucleic acid-based vaccines based on the Replikinsobserved in the emerging strain of cornaviruses are generated. Anemerging strain is identified as the strain of coronavirus having thehighest increase in concentration of Replikin sequences within thenucleocapsid sequence during the time period. Preferably, the peptide ornucleic acid vaccine is based on or includes any Replikin sequences thatare observed to be conserved in the strain. Conserved Replikins arepreferably those Replikin sequences which are present in thenucleocapsid protein sequence for about two years and preferably longer.The vaccines may include any combination of Replikin sequencesidentified in the emerging strain.

For vaccine production, the Replikin peptide or peptides identified asuseful for an effective vaccine are synthesized by any method, includingchemical synthesis and molecular biology techniques, including cloning,expression in a host cell and purification therefrom. The peptides arepreferably admixed with a pharmaceutically acceptable carrier in anamount determined to induce a therapeutic antibody reaction thereto.Generally, the dosage is about 0.1 mg to about 10 mg.

The coronavirus vaccine may be administered to a patient at any time ofthe year. Preferably, the coronavirus vaccine is administered once andis based on Replikin sequences observed to be present, and preferablyconserved, in the classes of coronavirus.

Example 5

Analysis of sequence data of isolates of Plasmodium falciparum antigensfor the presence and concentration of Replikins is carried out by visualscanning of sequences or through use of a computer program based on the3-point recognition method described herein. Isolates of Plasmodiumfalciparum are obtained and the amino acid sequence of the protein isobtained by any art known method, such as by sequencing the gene andderiving the protein sequence therefrom. Sequences are scanned for thepresence of Replikins, conservation of Replikins over time andconcentration of Replikins in each isolate. This information providesdata that are used to form the basis for anti-malarial peptide vaccinesor nucleic acid based vaccines.

Peptide vaccines or nucleic acid-based vaccines based on the Replikinsobserved in the malaria causing organism are generated. Preferably, thepeptide or nucleic acid vaccine is based on or includes any Replikinsequences that are observed to be present on a surface antigen of theorganism. The vaccines may include any combination of Replikin sequencesidentified in the malaria causing strain.

For vaccine production, the Replikin peptide or peptides identified asuseful for an effective vaccine are synthesized by any method, includingchemical synthesis and molecular biology techniques, including cloning,expression in a host cell and purification therefrom. The peptides arepreferably admixed with a pharmaceutically acceptable carrier in anamount determined to induce a therapeutic antibody reaction thereto.Generally, the dosage is about 0.1 mg to about 10 mg.

Then malaria vaccine is preferably administered to a patient in needthereof at any time during the year, and particularly prior to travel toa tropical environment.

Another aspect includes an antisense nucleic acid molecule complementaryto the coding strand of the gene or the mRNA encoding organism for thereplikins in organisms including, but not limited to, viruses,trypanosomes, bacteria, fungi, algae, amoeba, and plants, wherein saidantisense nucleic acid molecules is complementary to a nucleotidesequence of a replikin containing organism.

Example 6

Amino acid sequences of five short SARS Replikins found in nucleocapsid,spike, and envelope proteins of the SARS coronavirus were synthesizedand tested on rabbits to test immune response to Replikin sequences inthe SARS coronavirus. The following Replikin sequences were tested: (1)2003 Human SARS nucleocapsid (SEQ ID NO: 303); (2) 2003 Human SARS spikeprotein (SEQ ID NO: 304); (3) 2003 Human SARS spike protein (SEQ ID NO:305); 2003 Human SARS spike protein; (SEQ ID NO: 306); (4) 2003 SARSenvelope protein (SEQ ID NO: 307); and (5) 2003 Human SARS nucleocapsidprotein (SEQ ID NO: 308). Each synthesized peptide was injectedsubcutaneously into a rabbit. The tested rabbits produced measurablespecific antibody to each of the five sequences that bound at dilutionsof greater than 1 in 10,0000. The 21 amino acid SARS nucleocapsidreplikin antibody (SEQ. ID NO: 303) was demonstrated to bind atdilutions greater than 1 in 204,800. Because of previous unsuccessfulattempts by others to achieve with various small peptides a strongimmune response without the unwanted side effects obtained with a wholeprotein or the thousands of proteins or nucleic acids as in smallpoxvaccine, the ability of small synthetic replikin antigens to achievestrong immune responses was shown to be significant for the efficacy ofSARS vaccines.

Example 7

A 41 amino acid replikin sequenceKKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK (SEQ ID NO: 16)-KLH with theaddition of a key limpet hemocyanin adjuvant on the C-terminal end(denoted as -KLH was designated Vaccine V120304U2. The vaccine wasdesigned by the inventors from the 29 amino acid replikin Scaffold ofH5N1 “Bird Flu” Influenza Replikins labeled “2004H5N1 Vietnam, highlypathogenic” in Table 8 with the addition of two UTOPE units (KKKKHK)(SEQ ID NO: 459) on the C-terminal end of the H5N1 scaffold and anadditional adjuvant (key limpet hemocyanin adjuvant (denoted -KLH))covalently linked on the C-terminal end of the two UTOPE units. 100 ugof Vaccine V120304U2 was injected subcutaneously into rabbits andchickens. The antibody response was measured before vaccination and atfrom one week after injection to eight weeks after injection. Anantibody response was noted at one week and reached a peak in the thirdto fourth week after vaccination. Peak antibody responses ranged from adilution of 1:120,000 to a dilution of greater than 1:240,000. Antibodytiters were determined with an enzyme linked immunosorbent assay (ELISA)with Peptide-GGG (goat gamma globulin) bound in solid phase (0.1 ug/100ul/well) on high binding 96 well plates. The serum was first diluted 50fold and then further diluted in 2-fold serial dilutions. The ELISAtiter result was determined from the estimated dilution factor thatresulted from an optical density at 405 nm of 0.2 and derived fromnonlinear regression analysis of the serial dilution curve. Detectionwas obtained using a horse radish peroxidase conjugated secondaryantibody and ABTS substrate (ABTS is a registered trademark ofBoehringer Mannheim. GmbH). Results from tests on two chickens and tworabbits are provided in Table 11. Individual well results from the teston rabbit D4500 are provided in Table 12. In combination with theresults reported in Example 6, in a total of six tests of Replikinsequences for antibody responses in rabbit or chicken, all six sequencesprovided a measurable antibody response and have proved antigenic.

TABLE 11 Animal Bleed Day ELISA Titer Chickens injected with 100 μgV120304U2 on day 1. ELISA titer of antibody production on day 18 U0682Prior to <50 (Control) administration of vaccine u0682 18 daysafter >204,800 administration U0683 Prior to <50 (Control)administration of vaccine u0683 18 days after >204,800 administrationRabbits injected with 100 μg V120304U2 on day 1. ELISA titer of antibodyproduction on day 20 D4500 Prior to <50 (Control) vaccine administrationd4500 20 days after >204,800 administration D4501 Prior to 100 (Control)vaccine administration d4501 20 days after >204,800 administration

TABLE 12 Rabbits injected with 100 μg V120304U2 on day 1. OD450 resultsfor titers on days 7, 20 and 28 in individual wells Test Animal Day WellWell 1 Well 2 Well 3 Well 4 Well 5 6 d4500 Day 7 0.11 0.10 0.09 0.080.07 0.07 Day 20 0.49 0.38 0.23 0.19 0.22 0.17 Day 28 2.77 1.41 0.920.56 0.43 0.42 Well Well Well Well 7 Well 8 Well 9 10 11 12 d4500 Day 70.06 0.06 0.06 0.06 0.6  0.6  Day 20 0.02 0.16 0.17 0.15 0.19 0.28 Day28 0.17 0.14 0.12 0.11 0.11 0.10 Well Well 1 Well 2 Well 3 Well 4 Well 56 d4501 Day 7 0.25 0.18 0.15 0.11 0.09 0.08 Day 20 0.50 0.23 0.20 0.160.18 0.18 Day 28 1.75 0.84 0.61 0.50 0.34 0.35 Well Well Well Well 7Well 8 Well 9 10 11 12 d4501 Day 7 0.07 0.07 0.07 0.06 0.06 0.06 Day 200.16 0.18 0.16 0.17 0.17 0.25 Day 28 0.20 0.14 0.12 0.12 0.11 0.13

1. A method of identifying a series of peptide sequences as ReplikinScaffold peptides in a virus or organism comprising: isolating aplurality of peptides or polypeptides from one or more isolates of avirus, one or more isolates of a strain of virus, or one or moreisolates of an organism, analyzing the amino acid sequence of eachpeptide or polypeptide of the plurality of peptides, and identifying aseries of peptide sequences within the analyzed amino acid sequences asReplikin Scaffold peptides if each member of the series of identifiedpeptide sequences consists of about 16 to about 30 amino acids andcomprises (1) a terminal lysine and a lysine immediately adjacent tosaid terminal lysine; (2) a terminal histidine and a histidineimmediately adjacent to said terminal histidine, (3) a lysine withinabout 6 to about 10 amino acids from another lysine; and (4) at least 6%lysines, wherein the terminal lysines and terminal histidines thatdefine the Replikin Scaffold peptides within said series of ReplikinScaffold peptides are conserved across said different isolates of avirus, a strain of virus, or organism.
 2. The method of claim 1 furthercomprising: determining that said Replikin Scaffold peptides areisolated from different isolates of a virus, a strain of virus, or anorganism, wherein the lysine residues and the histidine residues thatdefine the Replikin Scaffold peptides are conserved across saiddifferent isolates of a virus, a strain of virus, or organism.
 3. Themethod of claim 2 further comprising: identifying a first ReplikinScaffold peptide and a second Replikin Scaffold peptide from among saidseries of identified peptide sequences, comparing said first ReplikinScaffold peptide to said second Replikin Scaffold peptide, and if saidsecond Replikin Scaffold peptide is unchanged from said first ReplikinScaffold peptide, choosing either the first Replikin Scaffold peptide orthe second Replikin Scaffold peptide to be comprised in a vaccine. 4.The method of claim 1, wherein isolating said plurality of peptides orpolypeptides comprises binding an antibody to at least one member ofsaid series of identified peptide sequences.
 5. The method of claim 1,wherein each member of the series of identified peptide sequencesconsists of 29 amino acids.
 6. The method of claim 1, wherein saidplurality of peptides or polypeptides are isolated from one or moreisolates of influenza virus.
 7. The method of claim 6, wherein said oneor more isolates of influenza virus includes at least one isolate froman H0N1, H1N1, H1N2, H2N2, H3N2, H5N1, H5N2, or H7N7 strain of influenzavirus.
 8. The method of claim 7, wherein said one or more isolates ofinfluenza virus is an H1N1 or H5N1 strain of influenza virus and atleast one of said plurality of peptides or polypeptides is isolated bybinding an antibody to at least one member of said series of identifiedpeptide sequences, and each member of the series of identified peptidesequences consists of 29 amino acids.